Modified proteins and methods of making and using same

ABSTRACT

Methods, compositions, systems, apparatuses and kits comprising modified proteins, particularly modified nucleic acid-binding proteins with altered buffering properties are provided. For example, in some embodiments, methods of forming modified proteins including one or more amino acid modifications to achieve desired pKa values are described. Furthermore, the invention provides methods for using such modified proteins in ion-producing reactions, such as ion-based nucleic acid sequencing reactions.

This application is a divisional of U.S. application Ser. No. 14/724,354filed May 28, 2015, now allowed, which is a Continuation of U.S.application Ser. No. 13/036,623, filed Feb. 28, 2011, now abandoned,which is a Continuation-in-Part of U.S. application Ser. No. 13/035,177,filed Feb. 25, 2011, now abandoned, which claims the benefit under 35U.S.C. § 119 of U.S. Provisional Application No. 61/308,863, filed Feb.26, 2010, all of which forgoing applications are herein incorporated byreference in their entireties. This application also claims the benefitof foreign priority to International Application Nos. PCT/US2011/026228,filed Feb. 25, 2011, and PCT/US2011/026468, filed Feb. 28, 2011, both ofwhich applications are incorporated herein by reference in theirentireties.

This application contains nucleotide sequence and/or amino acid sequencedisclosure in computer readable form and a written sequence listing, theentire contents of both of which are expressly incorporated by referencein their entirety as though fully set forth herein.

BACKGROUND

The direct detection of chemical moieties, such as ions, has tremendouspotential to simplify assay design and cost. For example, in someinstances, such techniques can reduce or eliminate the need for costlylabeling reagents; similarly, they can also eliminate the requirementfor complex detection steps that may otherwise be necessary. The utilityof such techniques can be illustrated by their application in the fieldof DNA sequence analysis, for which several chemical detection schemeshave been described. These include the detection of polymerase extensionby detecting physicochemical byproducts of the extension reaction, suchas pyrophosphate, hydrogen ion, charge transfer, heat, and the like, asdisclosed, for example, in Pourmand et al, Proc. Natl. Acad. Sci., 103:6466-6470 (2006); Purushothaman et al., IEEE ISCAS, IV-169-172; Rothberget al, U.S. Patent Publication No. 2009/0026082; Anderson et al, Sensorsand Actuators B Chem., 129: 79-86 (2008); Sakata et al., Angew. Chem.118:2283-2286 (2006); Esfandyapour et al., U.S. Patent Publication No.2008/01666727; and Sakurai et al., Anal. Chem. 64: 1996-1997 (1992).

Ion-based reactions, i.e., reactions involving the generation anddetection of ions, are widely performed. The use of direct ion detectionmethods to monitor the progress of such reactions can simplify manycurrent biological assays. For example, template-dependent nucleic acidsynthesis by a polymerase can be monitored by detecting hydrogen ionsthat are generated as natural byproducts of nucleotide incorporationscatalyzed by the polymerase. Ion-based sequencing (also referred to as“pH-based” nucleic acid sequencing) exploits the direct detection ofhydrogen ions produced as a byproduct of nucleotide incorporation. Inone exemplary system for ion-based sequencing, the nucleic acid to besequenced is captured in a microwell, and nucleotides are floated acrossthe well, one at a time, under nucleotide incorporation conditions. Thepolymerase incorporates the appropriate nucleotide into the growingstrand, and the hydrogen ion that is released changes the pH in thesolution, which is detected by an ion sensor. This technique does notrequire labeling of the nucleotides or expensive optical components, andallows for far more rapid completion of sequencing runs. Examples ofsuch ion-based nucleic acid sequencing methods including the Ion TorrentPGM™ sequencer (Life Technologies Corporation).

For ion-based reactions, including ion-based nucleic acid sequencing, itis important to detect as many released ions as possible in order toachieve as high a signal, and a correspondingly high signal to noiseratio, as possible. Obtaining sufficient signal can be challenging giventhe rapid diffusion of ions away from the reaction site, as well as thebuffering effects of other reaction components and the material of thecontainer wall. For example, the buffering effects of proteins in anion-based sequencing reaction can hinder the efficient detection ofhydrogen ions.

There is a therefore a need for methods and compositions for reducingthe buffering effects of protein reaction components, particularly foruse in ion-based nucleic acid sequencing methods and systems.

SUMMARY

In some embodiments, the disclosure relates generally to compositions,methods, systems, apparatuses and kits comprising modified proteinshaving reduced buffering capacities as compared to their unmodifiedcounterparts, as well as methods for making and using the modifiedproteins in a wide range of chemical reactions. In some embodiments, thedisclosure relates generally to methods, compositions, systems and kitscomprising modified proteins (e.g., nucleic acid binding proteins) foruse in ion-based nucleic acid sequence determination. In someembodiments, the disclosure relates generally to compositions, methods,systems, kits and apparatuses for carrying out a plurality of label-freeDNA sequencing reactions on a large-scale array of electronic sensors,for example field effect transistors (“FETs”).

In some embodiments, the disclosure relates generally to a modifiedprotein comprising one or more modifications that reduce the bufferingcapacity of the modified protein relative to the correspondingunmodified protein. Optionally, the one or more modifications reduce thebuffering capacity of the modified protein within the range of about pH4 to about pH 10. In some embodiments, the one or more modificationsreduce the buffering capacity of the modified protein relative to thecorresponding unmodified protein within the range of about pH 7 to aboutpH 9. In some embodiments, the one or more modifications include one ormore amino acid additions, substitutions, deletions, additions orchemical modifications. In some embodiments, the modified proteinincludes one or more amino acid substitutions, which are not included inthe unmodified protein.

In some embodiments, the disclosure relates generally to an isolatedprotein comprising one or more amino acid substitutions that reduce thebuffering capacity of the protein relative to the correspondingwild-type protein within the range of about pH 4 to about pH 10. In someembodiments, the one or more amino acid substitutions reduce thebuffering capacity of the protein relative to the correspondingwild-type protein within the range of about pH 7 to about pH 9.

In some embodiments, the modified protein is a nucleic acid bindingprotein. For example, the protein can have DNA- or RNA-binding activity.In various embodiments, the protein is selected from the groupconsisting of DNA polymerases, helicases, ligases, nucleases, singlestranded DNA binding proteins and polymerase accessory proteins.

In some embodiments, the one or more amino acid substitutionssubstantially reduce the buffering capacity of said protein within therange of about pH 7 to pH 9. In some embodiments, at least one of theone or more amino acid substitutions is a conservative amino acidsubstitution. In further embodiments, the at least one conservativeamino acid substitution is selected from the group consisting ofhistidine to arginine, glutamic acid to glutamine, aspartic acid toasparagine, lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, the protein is a DNA polymerase. In someembodiments, the disclosure relates generally to an isolated DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9. In some embodiments, the DNA polymerase is selected from the groupconsisting of an A family DNA polymerase; a B family DNA polymerase; amixed-type polymerase; an unclassified DNA polymerase and RT familypolymerase; and variants and derivatives thereof.

In some embodiments, the DNA polymerase is an A family DNA polymeraseselected from the group consisting of a Pol I-type DNA polymerase suchas E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, Bst DNA polymerase, Taq DNA polymerase, Platinum Taq DNApolymerase series, T7 DNA polymerase, and Tth DNA polymerase. In someembodiments, the DNA polymerase is Bst DNA polymerase. In otherembodiments, the DNA polymerase is E. coli DNA polymerase. In someembodiments, the DNA polymerase is the Klenow fragment of E. coli DNApolymerase. In some embodiments, the polymerase is Taq DNA polymerase.In some embodiments, the polymerase is T7 DNA polymerase.

In other embodiments, the DNA polymerase is a B family DNA polymeraseselected from the group consisting of Bst polymerase, Tli polymerase,Pfu polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase,KOD polymerase, Sac polymerase, Sso polymerase, Poc polymerase, Pabpolymerase, Mth polymerase, Pho polymerase, ES4 polymerase, VENTpolymerase, DEEPVENT polymerase, Therminator™ polymerase, phage Phi29polymerase, and phage B103 polymerase. In some embodiments, thepolymerase is KOD polymerase. In some embodiments, the polymerase isTherminator™ polymerase. In some embodiments, the polymerase is phagePhi29 DNA polymerase. In some embodiments the polymerase is phage B103polymerase, including, for example, the variants disclosed in U.S.Patent Publication No. 20110014612 which is incorporated by referenceherein.

In other embodiments, the DNA polymerase is a mixed-type polymeraseselected from the group consisting of EX-Taq polymerase, LA-Taqpolymerase, Expand polymerase series, and Hi-Fi polymerase. In yet otherembodiments, the DNA polymerase is an unclassified DNA polymeraseselected from the group consisting of Tbr polymerase, Tfl polymerase,Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tihpolymerase, and Tfi polymerase.

In other embodiments, the DNA polymerase is an RT polymerase selectedfrom the group consisting of HIV reverse transcriptase, M-MLV reversetranscriptase and AMV reverse transcriptase. In some embodiments, thepolymerase is HIV reverse transcriptase or a fragment thereof having DNApolymerase activity.

In some embodiments, the DNA polymerase is a Bst DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 2 or Table 3. Insome embodiments, the one or more conservative amino acid substitutionsare selected from the group consisting of H46R, H273R, H281R, E446Q,H473R, H528R, H572R and Y477F, the numbering of amino acid residuesbeing in accordance with that of SEQ ID NO: 1.

In some embodiments, the Bst DNA polymerase comprises one or moreconservative amino acid substitutions, wherein the one or more aminoacid substitutions includes a substitution of alanine at position 2 withMet, Asn, Gln, Leu, Ile, Phe, or Trp, the numbering of amino acidresidues being in accordance with that of SEQ ID NO: 1.

In some embodiments, the disclosure relates generally to an isolated BstDNA polymerase comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a variantthereof having one or more conservative amino acid substitutions. Insome embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 2. In some embodiments, the disclosure relatesgenerally to an isolated variant of a protein comprising the amino acidsequence shown in SEQ ID NO: 2, wherein the variant comprises an aminoacid sequence that is at least 95% identical to SEQ ID NO: 2.

In some embodiments, the disclosure relates generally to a Bst DNApolymerase comprising the amino acid sequence of SEQ ID NO: 3. In someembodiments, the disclosure relates generally to an isolated variant ofa protein comprising the amino acid sequence shown in SEQ ID NO: 3,wherein the variant comprises an amino acid sequence that is at least95% identical to SEQ ID NO: 3.

In some embodiments, the disclosure relates to a Bst DNA polymerasecomprising the amino acid sequence of SEQ ID NO: 4. In otherembodiments, the disclosure relates generally to an isolated variant ofa protein comprising the amino acid sequence shown in SEQ ID NO: 4,wherein the variant comprises an amino acid sequence that is at least95% identical to SEQ ID NO: 4.

In some embodiments, the DNA polymerase is a Therminator™ DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 5.

In some embodiments, the DNA polymerase is a KOD DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 6.

In some embodiments, the DNA polymerase is a B103 DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 7.

In other embodiments, the isolated protein comprising one or more aminoacid substitutions that reduce the buffering capacity of said proteinrelative to the corresponding wild-type protein within the range ofabout pH 4 to about pH 10 is a single stranded DNA binding protein(SSB).

In some embodiments, the SSB comprises one or more conservative aminoacid substitutions that substantially reduce its buffering capacitywithin the range of pH 7 to pH 9. In further embodiments, the one ormore conservative amino acid substitutions are selected from the groupconsisting of histidine to arginine, glutamic acid to glutamine,aspartic acid to asparagine, lysine to arginine, and tyrosine tophenylalanine. In some embodiments, the SSB is E. coli SSB. In furtherembodiments, the one or more conservative amino acid substitutions areof one or more amino acid residues shown in Table 4. In someembodiments, the SSB comprises the amino acid substitution K7R.

In some embodiments, the disclosure relates generally to an isolatedprotein comprising one or more amino acid substitutions that reduce thebuffering capacity of said protein relative to the correspondingwild-type protein within the range of about pH 4 to about pH 10, whereinat least one of the one or more amino acid substitutions includes asubstitution of an amino acid residue having a pKa within the range ofabout 4.0 to about 10.0 with another amino acid residue. In someembodiments, the pKa of the amino acid residue is a solution pKa of theamino acid residue. In other embodiments, the pKa of the amino acidresidue is a pKa of the amino acid residue in the context of thecorresponding wild-type protein.

In some embodiments, at least one of the one or more amino substitutionsincludes a substitution of an amino acid residue having a pKa of betweenabout 4.0 and about 10.0 with an amino acid residue having a pKa that isgreater than about 10.0 or less than about 4.0. In further embodimentsthe amino acid residue having a pKa that is greater than about 10.0 orless than about 4.0 is selected from the group consisting of: Arg, Asp,Gln, Lys, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp, Val andN-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 6.0 to about 8.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidmodifications includes a substitution of an amino acid residue having apKa of between about 6.0 and about 8.0 with an amino acid residue havinga pKa that is greater than about 8.0 or less than about 6.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue selectedfrom the group consisting of His, Glu, Asp, Tyr, and Lys with anotheramino acid residue.

In some embodiments, at least one of the one or more amino acidmodifications includes a substitution of an amino acid residue with analanine residue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue that isat least 30% solvent exposed in the corresponding wild-type protein withanother amino acid residue.

In some embodiments, the disclosure relates generally to an isolatedprotein comprising one or more chemical amino acid modifications thatreduce the buffering capacity of said protein relative to thecorresponding wild-type protein within the range of about pH 4 to aboutpH 10. In some embodiments, the one or more chemical amino acidmodifications includes a chemical modification of the N-terminal aminoacid. In some embodiments, the one or more chemical amino acidmodifications includes a chemical modification of an amino acid residueincluding a primary amine group with an amine-reactive agent. In someembodiments, the amine-reactive reagent includes an acylating agent oran activated ester.

In some embodiments, the disclosure relates generally to an isolatedpolymerase protein comprising one or more amino acid additions,substitutions, deletions or chemical modifications that reduce thebuffering capacity of said protein relative to the correspondingwild-type protein within the range of about pH 4 to about pH 10, whereinthe isolated protein retains polymerase activity.

In other embodiments, the disclosure relates generally to an isolatedprotein of any of the embodiments described above, further including anaffinity tag, a label, a chemical moiety, a radionuclide, an enzyme, afluorescent marker, a chemiluminescent marker, or a combination thereof.In other embodiments, the isolated protein is coupled to a polymer, asupport, or a sensor.

In other embodiments, the disclosure relates generally to an isolatednucleic acid that is at least 80% identical to a nucleic acid encoding aprotein of any of the embodiments described above. In furtherembodiments, the disclosure relates generally to a vector comprising theisolated nucleic acid. In further embodiments, the disclosure relatesgenerally to a host cell comprising the vector. In some embodiments, thehost cell is a bacterial cell. In some embodiments, the bacterial cellhas a reduced activity of methionine aminopeptidase (MAP) relative to acorresponding wild-type bacteria cell.

In other embodiments, the disclosure relates generally to a kitcomprising an isolated protein of any of the embodiments describedabove.

In some embodiments, the disclosure relates generally to an isolatedprotein comprising one or more amino acid additions, substitutions,deletions or chemical modifications that reduce the buffering capacityof said protein relative to the corresponding wild-type protein withinthe range of about pH 4 to about pH 10, wherein the one or more aminoacid substitutions substantially reduce the average number of sequencingerrors obtained from an ion-based sequencing reaction using saidprotein, relative to the number of sequencing errors obtained from anion-based sequencing reaction using the corresponding wild-type protein.

In some embodiments, the disclosure relates generally to an isolatednucleic acid binding polypeptide of claim comprising one or more aminoacid substitutions, deletions or chemical modifications that reduce thebuffering capacity of said protein relative to the correspondingwild-type protein within the range of about pH 4 to about pH 10, whereinthe one or more amino acid substitutions, deletions or chemicalmodifications increase the average length of sequencing reads having atleast 90% accuracy obtained from an ion-based sequencing reaction usingsaid, relative to average length of sequencing reads having 90% accuracyobtained from an ion-based sequencing reaction using the correspondingwild-type protein.

In some embodiments, the disclosure relates generally to a method forincorporating at least one nucleotide into a primer, comprising:contacting a nucleic acid duplex including a template nucleic acid and aprimer with a polymerase comprising one or more amino acidsubstitutions, deletions or chemical modifications that reduce thebuffering capacity of said polymerase relative to the correspondingwild-type polymerase within the range of about pH 4 to about pH 10 inthe presence of one or more nucleotides, and incorporating at least onenucleotide into the primer in a template-dependent fashion using saidpolymerase.

In some embodiments, the disclosure relates generally to methods forsequencing a nucleic acid using the modified proteins. In one exemplaryembodiment, the disclosure relates generally to a method for obtainingsequence information from a nucleic acid template, comprising:

(a) providing a template nucleic acid hybridized to a sequencing primerand bound to a polymerase, wherein the polymerase comprises one or moreamino acid substitutions that substantially reduce its bufferingcapacity within the range of about pH 4 to about pH 10;

(b) synthesizing a new nucleic acid strand by sequentially incorporatingone or more nucleotides at the 3′ end of the sequencing primer, whereina hydrogen ion byproduct is generated when the nucleotide iscomplementary to corresponding nucleotides in the template nucleic acid;and

(c) detecting the incorporation of the one or more nucleotides into thesequencing primer by detecting the release of hydrogen ions.

In some embodiments, the polymerase is a Bst DNA polymerase. In furtherembodiments, the one or more conservative amino acid substitutionswithin the polymerase are of one or more amino acid residues shown inTable 2 or Table 3. In further embodiments, the one or more conservativeamino acid substitutions are selected from the group consisting ofH341R, H568R, H576R, E741Q, H768R, H823R, H867R and Y772F. In someembodiments, the polymerase is selected from the group consisting of SEQID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a variant thereof having oneor more conservative amino acid substitutions.

In some embodiments, the method further comprises providing an SSB,wherein the SSB comprises one or more conservative amino acidsubstitutions that substantially remove its buffering capacity withinthe range of pH 7 to pH 9. In further embodiments, the SSB is E. coliSSB. In further embodiments, the one or more conservative amino acidsubstitutions in the SSB are of one or more amino acid residues shown inTable 4.

In another embodiment, the disclosure relates generally to a method forsequencing a nucleic acid, comprising: (a) disposing a plurality oftemplate nucleic acids into a plurality of reaction chambers, whereinone or more of the reaction chambers are in contact with a field effecttransistor (FET), and contacting at least one of the template nucleicacids with a polymerase including one or more conservative amino acidsubstitutions that substantially reduce its buffering capacity withinthe range of pH 7 to pH 9; (b) synthesizing a new nucleic acid strand bysequentially incorporating one or more nucleotides into a nucleic acidmolecule and generating one or more hydrogen ions as a byproduct of suchnucleotide incorporation; and (c) detecting the incorporation of the oneor more nucleotides by detecting the generation of the one or morehydrogen ions using the FET.

In some embodiments, the detecting includes detecting a change involtage and/or current at the at least one FET within the array inresponse to the generation of the one or more hydrogen ions.

In some embodiments, the FET is selected from the group consisting of:ion-sensitive FET (isFET) and chemically-sensitive FET (chemFET).

In some embodiments, the polymerase is Bst DNA polymerase. In furtherembodiments, the one or more conservative amino acid substitutionswithin the polymerase are of one or more amino acid residues shown inTable 2 or Table 3. In further embodiments, the one or more conservativeamino acid substitutions are selected from the group consisting ofH341R, H568R, H576R, E741Q, H768R, H823R, H867R and Y772F. In someembodiments, the polymerase is selected from the group consisting of SEQID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a variant thereof having oneor more conservative amino acid substitutions.

In some embodiments, the method further comprises providing an SSB,wherein the SSB comprises one or more conservative amino acidsubstitutions that substantially remove its buffering capacity withinthe range of pH 7 to pH 9. In further embodiments, the SSB is E. coliSSB. In further embodiments, the one or more conservative amino acidsubstitutions in the SSB are of one or more amino acid residues shown inTable 4.

In another embodiment, the disclosure relates generally to a method forsequencing a nucleic acid comprising:

(a) disposing a plurality of solid or semi-solid supports into aplurality of reaction chambers on a sensor array formed in asemiconductor substrate, at least one reaction chamber including apolymerase, a sequencing primer and a single solid or semi-solid supportattached to a plurality of template nucleic acids having at least 80%sequence identity to each other, wherein the polymerase comprises one ormore amino acid substitutions that substantially remove its bufferingcapacity within the range of pH 7 to pH 9, and each reaction chamber isin contact with or capacitively coupled to a sensor including a FETconfigured to provide at least one output representing the presence ofone or more hydrogen ions in the reaction chamber;

(b) introducing at least one nucleotide into the at least one reactionchamber and incorporating the at least one nucleotide into the primerusing the polymerase, thereby generating one or more hydrogen ionbyproducts;

(c) detecting the incorporation of the at least one nucleotides bydetecting the presence of the hydrogen ion byproducts.

In some embodiments, the method further includes repeating steps (a)through (c) until the nucleic acid is sequenced.

In some embodiments, the method further includes a step of washingunincorporated nucleotides from the at least one reaction chambers.

In some embodiments, the method further includes repeating steps (a)through (c) as well as the washing step until the nucleic acid issequenced.

In some embodiments, the polymerase is Bst DNA polymerase. In furtherembodiments, the one or more conservative amino acid substitutionswithin the polymerase are of one or more amino acid residues shown inTable 2 or Table 3. In further embodiments, the one or more conservativeamino acid substitutions are selected from the group consisting ofH341R, H568R, H576R, E741Q, H768R, H823R, H867R and Y772F. In someembodiments, the polymerase is selected from the group consisting of SEQID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a variant thereof having oneor more conservative amino acid substitutions.

In some embodiments, the method further comprises providing an SSB,wherein the SSB comprises one or more conservative amino acidsubstitutions that substantially remove its buffering capacity withinthe range of pH 7 to pH 9. In further embodiments, the SSB is E. coliSSB. In further embodiments, the one or more conservative amino acidsubstitutions in the SSB are of one or more amino acid residues shown inTable 4.

In a further embodiment, the disclosure relates generally to a methodfor reducing the buffering capacity of a protein used in a DNAsequencing or amplification reaction, comprising making one or moreamino acid substitutions in the protein sequence that substantiallyreduce the protein's buffering capacity within the range of about pH 4to about pH 10.

In some embodiments, the amino acid substitutions substantially reducethe protein's buffering capacity within the range of about pH 7 to aboutpH 9.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa in the range of about 4 to about 10 with an amino acid residuehaving a pKa less than about 4 or greater than about 10.

In some embodiments, the protein is a DNA- or RNA-binding protein.

In various embodiments, the protein is a DNA polymerase selected fromthe group consisting of a In some embodiments, the DNA polymerase is anA family DNA polymerase selected from the group consisting of a PolI-type DNA polymerase such as E. coli DNA polymerase, the Klenowfragment of E. coli DNA polymerase, Bst DNA polymerase, Taq DNApolymerase, Platinum Taq DNA polymerase series, T7 DNA polymerase, andTth DNA polymerase; a B family DNA polymerase selected from the groupconsisting of Bst polymerase, Tli polymerase, Pfu polymerase, Pfutubopolymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Sacpolymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, Pho polymerase, ES4 polymerase, VENT polymerase, DEEPVENTpolymerase, phage Phi29 polymerase, and phage B103 polymerase; amixed-type polymerase selected from the group consisting of EX-Taqpolymerase, LA-Taq polymerase, Expand polymerase series, and Hi-Fipolymerase; an unclassified DNA polymerase selected from the groupconsisting of Tbr polymerase, Tfl polymerase, Tru polymerase, Tacpolymerase, Tne polymerase, Tma polymerase, Tih polymerase, and Tfipolymerase; and an RT polymerase selected from the group consisting ofHIV reverse transcriptase, M-MLV reverse transcriptase and AMV reversetranscriptase.

In some embodiments, the polymerase is Bst DNA polymerase. In furtherembodiments, the one or more conservative amino acid substitutionswithin the polymerase are of one or more amino acid residues shown inTable 2 or Table 3. In further embodiments, the one or more conservativeamino acid substitutions are selected from the group consisting ofH341R, H568R, H576R, E741Q, H768R, H823R, H867R and Y772F. In someembodiments, the polymerase is selected from the group consisting of SEQID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a variant thereof having oneor more conservative amino acid substitutions.

In some embodiments, the method further comprises providing an SSB,wherein the SSB comprises one or more conservative amino acidsubstitutions that substantially remove its buffering capacity withinthe range of pH 7 to pH 9. In further embodiments, the SSB is E. coliSSB. In further embodiments, the one or more conservative amino acidsubstitutions in the SSB are of one or more amino acid residues shown inTable 2.

In a further embodiment, the disclosure relates generally to a method ofdetecting a nucleotide incorporation, comprising:

(a) performing a nucleotide incorporation using a modified polymeraseand generating one or more hydrogen ions as a by-product of thenucleotide incorporation, where the modified polymerase includes one ormore amino acid substitutions that reduce the buffering capacity of themodified polymerase relative to the unmodified polymerase; and

(b) detecting the presence of the one or more hydrogen ions generated asa by-product of the nucleotide incorporation, thereby detecting thenucleotide incorporation.

In some embodiment, the modified polymerase includes one or more aminoacid substitutions that reduce the buffering capacity of the modifiedpolymerase within the pH range of about 4 to about 10 relative to theunmodified polymerase.

In some embodiments the detecting comprises using an ion-sensitive fieldeffect transistor (ISFET). In some embodiments the ISFET is in an ISFETarray.

In some embodiments the reaction is performed in a reaction chambercomprising or capacitively coupled to a chemFET.

In some embodiments, the reaction is in the presence of an agent thatalters the value of a pKa of at least an amino acid in the modifiedpolypeptide from within the range of about 6 to about 8 to less thanabout 6 or greater than about 8. In some embodiments, the agent is aphospholipid, a sulfonic acid surfactant, a polyanionic electrolyte or asalt thereof, a polycationic electrolyte or a salt thereof, tetramethylammonium or a salt thereof, or a combination thereof.

In some embodiments, the method further comprises repeating steps a)-b).

In a further aspect, the disclosure relates generally to a method ofdetecting a change in ion concentration during a chemical reaction,comprising:

(a) performing a chemical reaction in the presence of a modifiedpolypeptide having one or more amino acid substitutions, wherein theconcentration of at least one type of ion changes during the course ofthe chemical reaction; and

(b) detecting a signal indicating the change in ion concentration,wherein the signal is increased relative to a signal that is detectedfrom a chemical reaction performed in the presence of the unmodifiedpolypeptide but under otherwise identical reaction conditions.

In some embodiments the modified polypeptide includes one or more aminoacid substitutions that reduce the buffering capacity of the modifiedpolypeptide relative to the unmodified polypeptide.

In some embodiments at least one of the one or more amino acidsubstitutions includes the substitution of an amino acid residue havinga pKa of between about 4 and 10 with another amino acid residue having apKa less than about 4 or greater than about 10.

In some embodiments the modified polypeptide is a modified polymerase,and the chemical reaction includes a nucleotide incorporation.

In some embodiments the detecting comprises using a chemFET. In someembodiments the chemFET is an ISFET. In some embodiments, the ISFET isin an ISFET array.

In some embodiments, the chemical reaction is performed in a reactionchamber comprising or capacitively coupled to a chemFET.

In some embodiments, the chemical reaction is in the presence of anagent that alters the value of a pKa of at least an amino acid in themodified polypeptide from within the range of about 6 to about 8 to lessthan about 6 or greater than about 8. In some embodiments, the agent isa phospholipid, a sulfonic acid surfactant, a polyanionic electrolyte ora salt thereof, a polycationic electrolyte or a salt thereof,tetramethyl ammonium or a salt thereof, or a combination thereof.

In some embodiments, the at least one type of ion is hydrogen ion.

In some embodiments, the reaction is an enzymatic reaction or a bindingreaction.

In some embodiments, the chemical reaction is an enzymatic reaction. Insome embodiments the enzymatic reaction is a nucleotide incorporationreaction.

In some embodiments, the method further comprises repeating steps a)-b).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid sequence of the large fragment of the BstDNA polymerase. The conserved polymerase motifs are highlighted andunderlined, and the invariant residues within these motifs are shown inbold.

FIG. 2 shows a sequence alignment of the amino acid sequences of thebacteriophage B103 DNA polymerase (SEQ ID NO:8) and the Phi29 DNApolymerase (SEQ ID NO:18).

FIG. 3 shows the 50Q17 vs. Keypass for a sequencing reaction using theunmodified Bst DNA polymerase having the amino acid sequence of SEQ IDNO: 1 (“manta”) and the modified Bst DNA polymerase having the aminoacid sequence of SEQ ID NO: 2 (“ion”).

FIG. 4 shows the 100Q17 vs. Keypass for a sequencing reaction using theunmodified Bst DNA polymerase having the amino acid sequence of SEQ IDNO: 1 (“manta”) and the modified Bst DNA polymerase having the aminoacid sequence of SEQ ID NO: 2 (“ion”).

FIG. 5 shows the Carryforward vs. 50Q17/Keypass for a sequencingreaction using the unmodified Bst DNA polymerase having the amino acidsequence of SEQ ID NO: 1 (“manta”) and the modified Bst DNA polymerasehaving the amino acid sequence of SEQ ID NO: 2 (“ion”).

FIG. 6 shows some exemplary data obtained from an ion-based sequencingreaction using a modified polymerase having the amino acid sequence ofSEQ ID NO: 2 (“ion”) and a reference polymerase having the amino acidsequence of SEQ ID NO: 1. The first four runs (Run Nos. 1-4) wereperformed using a modified polymerase having the amino acid sequence ofSEQ ID NO: 1, while the last three runs (Run Nos. 5-7) were performedusing the reference (unmodified) Bst polymerase having the amino acidsequence of SEQ ID NO: 1.

DETAILED DESCRIPTION

All patents, patent applications, published applications, treatises andother publications referred to herein, both supra and infra, areincorporated by reference in their entirety. If a definition and/ordescription is explicitly or implicitly set forth herein that iscontrary to or otherwise inconsistent with any definition set forth inthe patents, patent applications, published applications, and otherpublications that are herein incorporated by reference, the definitionand/or description set forth herein prevails over the definition that isincorporated by reference.

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the arts to which thisdisclosure pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art.

Throughout this disclosure, various amino acid mutations, including, forexample, amino acid substitutions are referenced using the amino acidsingle letter code, and indicating the position of the residue within areference amino acid sequence. In the case of amino acid substitutions,the identity of the substituent is also indicated using the amino acidsingle letter code. For example, a reference to the hypothetical aminoacid substitution “D166A, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 7” indicates an amino acid substitutionwherein an alanine (A) residue is substituted for the normally occurringaspartic acid (D) residue at amino acid position 166 of the amino acidsequence of SEQ ID NO: 7. Many of the amino acid sequences disclosedherein begin with a methionine residue (“M”), which is typicallyintroduced at the beginning of nucleic acid sequences encoding peptidesdesired to be expressed in bacterial host cells. However, it is to beunderstood that the disclosure also encompasses all such amino acidsequences beginning from the second amino acid residue onwards, withoutthe inclusion of the first methionine residue.

As used herein, the term “amino acid” and its variants include withoutexception naturally occurring and synthetic amino acids, as well asamino acid analogs and amino acid mimetics that function in a mannersimilar to the naturally occurring amino acids. Naturally occurringamino acids are those encoded by the genetic code, includingselenomethionine, as well as those amino acids that are modified afterincorporation into a polypeptide, e.g., hydroxyproline,γ-carboxyglutamate, O-phosphoserine, and cystine. “Amino acid analog”refers to compounds that have the same basis chemical structure as anaturally occurring amino acid, i.e., an α-carbon that is bound by ahydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetic” refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. Amino acids may bereferred to herein by either their commonly known three letter symbolsor by their one letter symbols.

As used herein, the term “percent (%) amino acid sequence identity” andits variants, when used in reference to one or more polypeptidesequences, is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

As used herein, the terms “polynucleotide” and “oligonucleotide” andtheir variants, which can be used interchangeably, include a linearpolymer of nucleotide monomers. Monomers making up polynucleotides andoligonucleotides are capable of specifically binding to a naturalpolynucleotide by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing, base stacking,Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Suchmonomers and their internucleosidic linkages may be naturally occurringor may be analogs thereof, e.g. naturally occurring or non-naturallyoccurring analogs. Non-naturally occurring analogs may include PNAs,phosphorothioate internucleosidic linkages, bases containing linkinggroups permitting the attachment of labels, such as fluorophores, orhaptens, and the like. Whenever the use of an oligonucleotide orpolynucleotide requires enzymatic processing, such as extension by apolymerase, ligation by a ligase, or the like, one of ordinary skillwould understand that oligonucleotides or polynucleotides in thoseinstances would not contain certain analogs of internucleosidiclinkages, sugar moieties, or bases at any or some positions. Whenever apolynucleotide or oligonucleotide is represented by a sequence ofletters (upper or lower case), such as “ATGCCTG,” it will be understoodthat the nucleotides are in 5′ to 3′ order from left to right and that“A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotesdeoxyguanosine, and “T” denotes thymidine, “I” denotes deoxyinosine, “U”denotes uridine, unless otherwise indicated or obvious from context.Unless otherwise noted the terminology and atom numbering conventionswill follow those disclosed in Strachan and Read, Human MolecularGenetics 2 (Wiley-Liss, New York, 1999). Usually polynucleotidescomprise the four natural nucleosides (e.g. deoxyadenosine,deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribosecounterparts for RNA) linked by phosphodiester linkages; however, theymay also comprise non-natural nucleotide analogs, e.g. includingmodified bases, sugars, or internucleosidic linkages. It is clear tothose skilled in the art that where an enzyme has specificoligonucleotide or polynucleotide substrate requirements for activity,e.g. single stranded DNA, RNA/DNA duplex, or the like, then selection ofappropriate composition for the oligonucleotide or polynucleotidesubstrates is well within the knowledge of one of ordinary skill,especially with guidance from treatises, such as Sambrook et al,Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, NewYork, 1989), and like references.

As used herein, the term “primer” and its variants include anypolynucleotide, either natural or synthetic that is capable, uponforming a duplex with a polynucleotide template, of acting as a point ofinitiation of nucleic acid synthesis and being extended from its 3′ endalong the template so that an extended duplex is formed. The primer neednot exhibit 100% complementarity with the polynucleotide template, andmay hybridize only partially with the nucleic acid template in order tobe capable of initiating nucleic acid synthesis. Extension of a primeris usually carried out with a nucleic acid polymerase, such as a DNA orRNA polymerase. The sequence of nucleotides added in the extensionprocess is typically determined by the sequence of the templatepolynucleotide. In some embodiments, template-dependent nucleotideincorporation proceeds according to standard base-pairing paradigms,such as the Watson-Crick paradigm wherein A nucleotides typically pairwith T or U, and G typically pairs with C. In other embodiments, theorder of nucleotide incorporation can be governed by any other,non-traditional base pairing paradigm.

In some embodiments, primers can be extended by a DNA polymerase or anRNA polymerase. The primers can optionally have a length in the range offrom 14 to 40 nucleotides, or in the range of from 18 to 36 nucleotides.Primers can be employed in a variety of nucleic amplification reactions,for example, linear amplification reactions using a single primer, orpolymerase chain reactions, employing two or more primers. Guidance forselecting the lengths and sequences of primers for particularapplications is well known to those of ordinary skill in the art, asevidenced by the following references that are incorporated byreference: Dieffenbach, editor, PCR Primer: A Laboratory Manual, 2^(nd)Edition (Cold Spring Harbor Press, New York, 2003).

In some embodiments, the disclosure relates generally to modifiedproteins having altered, e.g., increased or reduced, bufferingcapacities. Such modified proteins have utility, for example, inion-sensitive reactions that involve the detection and/or quantificationof ion byproducts (e.g., H+ ion or OH− ion byproducts) in the presenceof the protein. Detection of such ion byproducts can be complicated bythe intrinsic buffering capacity of the protein, which may result inbinding of the protein to one or more ion byproducts, thereby reducingthe number of ion byproducts available for detection and/orquantification. Use of a modified protein having a reduced bufferingcapacity can reduce or eliminate such complications, thereby increasingthe amount of ions available for detection and/or quantification.

As used herein, the terms “buffering capacity” and its variants refer,among other things, to the ability of a composition to bind a particulartype of ion (typically H+ ions or OH− ions) in solution. The greater thedegree to which the particular type ion can be, or is, bound bycomposition, the higher the buffering capacity of the composition. Forexample, in some embodiments the buffering capacity of a particularcomposition (e.g., a protein) can be defined as the total mass of H+ions (in moles or millimoles) absorbed by a unit mass (e.g., moles ormillimole) of the composition (e.g., protein) under defined reactionconditions. Proteins typically exhibit a buffering capacity, which canbe influenced by the number and type of side chains in the amino acidresidues within the protein. In other embodiments, the bufferingcapacity of a composition can be measured as the composition'sresistance to pH change upon addition of acid or base in solution. Insuch embodiments, the buffering capacity of a protein may be determinedby performing a conventional pH titration on a known quantity of thecandidate protein and determining the characteristics of the titrationcurve in the pH range of interest.

Typically, the modified proteins include one or more amino acidmodifications, while the reference protein lacks at least one of thesemodifications. In some embodiments, the one or more amino acidmodifications can alter (e.g., increase or reduce) the bufferingcapacity of the modified protein relative to the buffering capacity ofthe reference protein. The one or more modifications can include one ormore amino acid substitutions, deletions, additions or chemicalmodifications. In one typical embodiment, the modified protein and thereference proteins are polymerases.

The reference protein can be any suitable protein whose bufferingcapacity can be measured and compared to the activity of a modifiedprotein of the present disclosure. In some embodiments, the modifiedproteins include one or more amino acid modifications, while thereference protein lacks at least one of these modifications but isotherwise identical to the modified protein. In some embodiments, thereference protein can be the unmodified counterpart of the modifiedprotein; for example, the reference protein can be the wild-typecounterpart of the modified protein. In some embodiments, the referenceprotein is a naturally occurring protein.

In some embodiments, the modified proteins of the disclosure can exhibitreduced buffering capacity in a desired pH range. For example, in someembodiments, the reduced buffering capacity is manifested, or can beobserved, within the range of from about pH 4 to about pH 10, or fromabout pH 5.5 to about pH 9.5, or from about pH 7 to about pH 9.

In some embodiments, the modified proteins of the disclosure can exhibitreduced buffering capacity such that relative small changes inconcentration of hydrogen ions in a solution comprising the protein willproduce relatively large changes in measured pH; such changes aretypically greater than the changes in measured pH observed using theunmodified counterpart of the modified protein.

In some embodiments, the disclosure relates generally to modifiedproteins that have reduced buffering capacity relative to thecorresponding unmodified (e.g., wild type protein).

In some embodiments, the modified protein comprises one or more aminoacid substitutions that reduce the buffering capacity of the modifiedprotein relative to the corresponding wild-type protein within the rangeof about pH 4 to about pH 10, wherein at least one of the one or moreamino acid substitutions includes a substitution of an amino acidresidue having a pKa within the range of about 4.0 to about 10.0 withanother amino acid residue. In some embodiments, the pKa of the aminoacid residue is a solution pKa of the amino acid residue. In otherembodiments, the pKa of the amino acid residue is a pKa of the aminoacid residue in the context of the corresponding wild-type protein.

In some embodiments, at least one of the one or more amino substitutionsincludes a substitution of an amino acid residue having a pKa of betweenabout 4.0 and about 10.0 with an amino acid residue having a pKa that isgreater than about 10.0 or less than about 4.0. In further embodimentsthe amino acid residue having a pKa that is greater than about 10.0 orless than about 4.0 is selected from the group consisting of: Arg, Asp,Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp, Val andN-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 6.0 to about 8.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidmodifications includes a substitution of an amino acid residue having apKa of between about 6.0 and about 8.0 with an amino acid residue havinga pKa that is greater than about 8.0 or less than about 6.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue selectedfrom the group consisting of His, Glu, Asp, Tyr, and Lys with anotheramino acid residue.

In some embodiments, at least one of the one or more amino acidmodifications includes a substitution of an amino acid residue with analanine residue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue that isat least 30% solvent exposed in the corresponding wild-type protein withanother amino acid residue.

In some embodiments of the method, the polymerase comprises one or morechemical amino acid modifications that reduce the buffering capacity ofsaid protein relative to the corresponding wild-type protein within therange of about pH 4 to about pH 10. In some embodiments, the one or morechemical amino acid modifications includes a chemical modification ofthe N-terminal amino acid. In some embodiments, the one or more chemicalamino acid modifications includes a chemical modification of an aminoacid residue including a primary amine group with an amine-reactiveagent. In some embodiments, the amine-reactive reagent includes anacylating agent or an activated ester.

In some embodiments, at least one of the one or more amino acidmodifications of the modified protein includes a substitution of anamino acid residue having a pKa of between about 4.0 and about 10.0 withan amino acid residue having a pKa that is greater than about 10.0 orless than about 4.0. In some embodiments, the amino acid residue havinga pKa that is greater than about 10.0 or less than about 4.0 is selectedfrom the group consisting of: Arg, Asp, Gln, ly, Ile, Leu, Norleucine(Nle), Met, Phe, Ser, Thr, Trp, Val and N-terminal Formylmethionine(N-fMet).

In some embodiments, at least one of the one or more amino acidmodifications of the modified protein includes a substitution of anamino acid residue having a pKa within the range of about 6.0 to about8.0 with another amino acid residue. The pKa can optionally be thesolution pKa or the whole-protein pKa of the amino acid residue.

In some embodiments, at least one of the one or more amino acidmodifications of the modified protein includes a substitution of anamino acid residue having a pKa of between about 6.0 and about 8.0 withan amino acid residue having a pKa that is greater than about 8.0 orless than about 6.0. In some embodiments, the amino acid to besubstituted is His, Glu, Asp, Tyr or Lys.

In some embodiments, the modified protein includes at least oneconservative amino acid substitution selected from the group consistingof His to Arg, Glu to Gln, Asp to Asn, Lys to Arg, and Tyr to Phe.

In some embodiments, at least one of the one or more amino acidmodifications includes a substitution of an amino acid with an alanineresidue.

In some embodiments, at least one of the one or more amino acidmodifications includes a deletion of an amino acid. In some embodiments,at least one of the one or more deleted amino acids includes an aminoacid residue having a pKa of between about 4.0 and about 10.0. In someembodiments, the amino acid residue having a pKa that is greater thanabout 10.0 or less than about 4.0 is selected from the group consistingof: Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr,Trp, Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more deleted amino acidsis an amino acid residue having a pKa within the range of about 6.0 toabout 8.0 with another amino acid residue. In some embodiments, thedeleted amino acid is His, Glu, Asp, Tyr or Lys.

The buffering capacity of a composition can be measured in various ways.In some embodiments, the buffering capacity of a composition can bemeasured in terms of the effect of the composition upon theconcentrations of one or more types of ions in a solution. For example,the buffering capacity of a protein can be measured by measuring the H+ion concentration in an aqueous solution that includes the protein. Thesolution can optionally include other components such as salts,nucleotides, and the like, which may or may not independently havebuffering capacity. In other embodiments, the buffering capacity of acomposition can be measured in terms of the amount of strong acid orbase required to change the pH of a composition a given amount. VanSlyke (J. Biol. Chem. 52: 525-570 (1922)) provided a widely usedconventional quantitative measure of buffering capacity, according towhich, for a solution, buffering capacity is expressed as the amount ofstrong acid or base required to change the pH of one liter of thesolution by one pH unit under standard conditions of temperature andpressure.

In some embodiments, the modified protein includes one or moremodifications that increase or decrease the buffering capacity of themodified protein for a particular ion (e.g., H+ ions or OH− ions). Forexample, the one or more modifications can reduce the buffering capacityof the modified protein for hydrogen ions (e.g., H⁺ ions). Optionally,the reduction in buffering capacity for H⁺ ions is manifested over aparticular pH range. In some embodiments, the one or more modificationscan increase the intensity of the signal detected in an H+ ion-sensitivereaction, where the signal indicates the amount of H+ ions present inthe ion-sensitive reaction. The H+ ion-sensitive reaction can be anucleic acid sequencing reaction, wherein one or more H+ ions arereleased as a byproduct of nucleotide incorporation by a polymerase. Insome embodiments, the signal indicating the amount of H+ ions present inthe ion-sensitive reaction can be generated using a suitablefield-effect transistor (FET), for example a chemFET or an isFET, asdescribed in further detail below.

In some embodiments, the reduction in buffering capacity of the modifiedprotein is determined by measuring the buffering capacity of themodified protein for a particular ion of interest (e.g., hydrogen ions),measuring the buffering capacity of a reference protein for the sameion, and comparing the buffering capacity of the modified protein to thebuffering capacity of the reference protein. For example, an increase ordecrease in the buffering capacity for hydrogen ions of particularmodified protein can be determined by measuring the H+ ion concentrationin a solution (“test solution”) including the modified protein andcomparing it to the H+ ion concentration in a solution (“referencesolution”) including the reference protein. In some embodiments, themodified protein is a mutant polymerase exhibiting reduced bufferingcapacity for H+ ions relative to a reference polymerase (for example,the corresponding wild-type version of the mutant polymerase) and thereduction in buffering capacity is determined by measuring the amount ofH+ ions detected in a polymerase reaction solution including themodified protein, which is then compared to the amount of H+ ionsmeasured in a polymerase reaction solution including the referencepolymerase in lieu of the modified polymerase. In some embodiments, theamount of H+ ions present in test and/or reference solutions is measuredusing a FET, for example a chemFET or an isFET.

As used herein, the terms “buffer”, “buffering agent” and their variantsinclude any substance that binds H+ ions or OH− ions in solution.Typically, such buffers or buffering agents prevent or reduce any changein the acidity of a solution when an acid or base is added to thesolution. For example, aqueous buffers typically scavenge H+ or OH− ionsin an aqueous solution. Buffers can in some embodiments comprising aconjugate acid-base pair that scavenges the H+ or OH− ions in thesolution. Any buffer will have a pKa value, the pH at which the bufferhas its maximum buffering capacity. The buffering action of the buffertypically manifests over a pH range including from at least about one ortwo pH units below its pKa to at least about one or two pH units aboveits pKa value.

As used herein, the terms “non-buffering” and “bufferless” and theirvariants, when used in reference to a protein, refer to a protein havingreduced, slight or no buffering capacity for a given type of ion (e.g.,H⁺ ions or OH⁻ ions) in a given pH range. Such proteins can be usefulfor ion-based reactions requiring detection of the ion type in thepresence of the protein. For example, a small change in the ionconcentration can increase the amplitude of a signal indicating the ionconcentration in a reaction including the modified protein, relative toa reaction including a protein having a higher buffering capacity.Typically, such non-buffering proteins include modified proteins thatexhibit reduced buffering capacities for a given type of ion (e.g., H⁺ions, OH⁻ ions) relative to their unmodified counterparts.

In some embodiments, the disclosure relates generally to use of modifiedproteins having reduced buffering capacity in nucleic acid sequencing oramplification reactions. The modified proteins may include both DNA andRNA binding proteins. Such proteins may or may not be directly involvedin nucleic acid extension in such reactions, and may include nucleicacid polymerases, single stranded DNA binding proteins, ligases,helicases, nucleases, and polymerase accessory proteins. In someembodiments, the modified protein having altered (e.g., increased ordecreased) buffering capacity is a polymerase. The polymerase can be aDNA polymerase or an RNA polymerase. As used herein, the term“polymerase” and its variants comprise any enzyme that can catalyze thepolymerization of nucleotides (including analogs thereof) into a nucleicacid strand. Typically but not necessarily such nucleotidepolymerization can occur in a template-dependent fashion.

In some embodiments, the modified polymerases can be used to sequencenucleic acids in an ion-based sequencing reaction. One exemplary systemis the Ion Torrent PGM™ sequencer (Life Technologies), which is anion-based sequencing system that sequences nucleic acid templates bydetecting ions produced as a byproduct of nucleotide incorporation.Typically, hydrogen ions are released as byproducts of nucleotideincorporations occurring during template-dependent nucleic acidsynthesis by a polymerase. The Ion Torrent PGM™ sequencer detects thenucleotide incorporations by detecting the hydrogen ion byproducts ofthe nucleotide incorporations. The Ion Torrent PGM™ sequencer includes aplurality of nucleic acid templates to be sequenced, each templatedisposed within a respective sequencing reaction well in an array. Thewells of the array are each coupled to at least one ion sensor that candetect the release of H⁺ ions or changes in solution pH produced as abyproduct of nucleotide incorporation. The ion sensor comprises a fieldeffect transistor (FET) coupled to an ion-sensitive detection layer thatcan sense the presence of H⁺ ions or changes in solution pH. The ionsensor provides output signals indicative of nucleotide incorporationwhich can be represented as voltage changes whose magnitude correlateswith the H⁺ ion concentration in a respective well or reaction chamber.Different nucleotide types are flowed serially into the reactionchamber, and are incorporated by the polymerase into an extending primer(or polymerization site) in an order determined by the sequence of thetemplate. Each nucleotide incorporation is accompanied by the release ofH⁺ ions in the reaction well, along with a concomitant change in thelocalized pH. The release of H⁺ ions is registered by the FET of thesensor, which produces signals indicating the occurrence of thenucleotide incorporation. Nucleotides that are not incorporated during aparticular nucleotide flow will not produce signals. The amplitude ofthe signals from the FET may also be correlated with the number ofnucleotides of a particular type incorporated into the extending nucleicacid molecule thereby permitting homopolymer regions to be resolved.Thus, during a run of the sequencer multiple nucleotide flows into thereaction chamber along with incorporation monitoring across amultiplicity of wells or reaction chambers permit the instrument toresolve the sequence of many nucleic acid templates simultaneously.Further details regarding the compositions, design and operation of theIon Torrent PGM™ sequencer can be found, for example, in U.S. patentapplication Ser. No. 12/002,781, now published as U.S. PatentPublication No. 2009/0026082; U.S. patent application Ser. No.12/474,897, now published as U.S. Patent Publication No. 2010/0137143;and U.S. patent application Ser. No. 12/492,844, now published as U.S.Patent Publication No. 2010/0282617, all of which applications areincorporated by reference herein in their entireties.

In some embodiments, the disclosure relates generally to use of themodified proteins of the present disclosure in methods of ion-basedsequencing. Typically, the modified protein is a polymerase includingone or more amino acid substitutions that reduce the buffering capacityof the polymerase for H⁺ ions relative to an unmodified (e.g.,wild-type) counterpart under sequencing reaction conditions. Use of suchmodified polymerases having reduced buffering capacities in ion-basedsequencing reactions is particularly advantageous because the intrinsicbuffering capacity of the polymerase can reduce the number of H⁺ ionsavailable for detection by the FET sensor and thus reduce the magnitudeof the signal, as well as decrease the signal-to-noise ratio, associatedwith nucleotide incorporation. Such signal interference resulting fromthe polymerase's buffering properties can result in sequencing errorsand reduce the average error-free read length in an ion-based sequencingsystem.

In some embodiments, use of modified polymerases having reducedbuffering capacities reduces or eliminates such effects, therebyincreasing the magnitude of the voltage change associated with aparticular nucleotide incorporation in an ion-based sequencing system.

In some embodiments, use of modified polymerases having reducedbuffering capacities for H⁺ ions can increase the magnitude of thesignal indicating a nucleotide incorporation and/or decrease thesignal-to-noise ratio for the signal obtained in an ion-based sequencingsystem.

In some embodiments, use of the modified polymerases having reducedbuffering capacities for H⁺ ions increases the average length oferror-free sequencing reads having an error rate of no greater than 1error per 100 nucleotides in an ion-based sequencing system.

In some embodiments, use of the modified polymerases having reducedbuffering capacities for H⁺ ions decreases the average sequencing errorrate in an ion-based sequencing system.

In some embodiments, the modified polymerases exhibit reduced bufferingcapacities in for H⁺ ions in a pH range of from about pH 4 to about pH10. Sequencing reactions are typically performed at pH values of fromabout pH 6 to about pH 8. In some embodiments, the modified polymerasesexhibit reduced buffering capacities in for H⁺ ions in a pH range offrom about pH 6 to about pH 8.

In some embodiments, the polymerases of the present disclosure caninclude without limitation naturally occurring polymerases and anysubunits and truncations thereof, mutant polymerases, variantpolymerases, recombinant, fusion or otherwise engineered polymerases,chemically modified polymerases, synthetic molecules or assemblies,and/or any analogs, derivatives or fragments thereof that retain theability to catalyze such polymerization. Optionally, the polymerase canbe a mutant polymerase comprising one or more mutations involving thereplacement of one or more amino acids with other amino acids, theinsertion or deletion of one or more amino acids from the polymerase, orthe linkage of parts of two or more polymerases. Typically, thepolymerase comprises one or more active sites at which nucleotidebinding and/or catalysis of nucleotide polymerization can occur. Someexemplary polymerases include without limitation DNA polymerases (suchas for example Phi-29-type DNA polymerase, reverse transcriptases and E.coli DNA polymerase) and RNA polymerases. In some embodiments, themodified polymerase can be a fusion protein comprising at least twoportions linked to each other, where the first portion comprises a firstpolypeptide that can catalyze the polymerization of nucleotides into anucleic acid strand, where the first portion is linked to a secondportion that comprises a second polypeptide, such as, for example, areporter enzyme or a processivity-enhancing domain One exemplaryembodiment of such a polymerase is Phusion® DNA polymerase (New EnglandBiolabs), which comprises a Pyrococcus-like polymerase fused to aprocessivity-enhancing domain as described, for example, in U.S. Pat.No. 6,627,424.

In some embodiments, the modified polymerase is derived from a known DNApolymerase. The DNA polymerases have been classified into sevendifferent families, based upon both amino acid sequence comparisons andthree-dimensional structure analyses. The DNA polymerase I (pol I) ortype A polymerase family includes the repair polymerases E. coli DNA polI, Thermus aquaticus pol I, and Bacillus stearothermophilus pol I,replicative DNA polymerases from some bacteriophages (T3, T5 and T7) andeukaryotic mitochondrial DNA polymerases. The DNA polymerase α (pol α)or type B polymerase family includes all eukaryotic replicating DNApolymerases as well as archaebacterial DNA polymerases, viral DNApolymerases, DNA polymerases encoded in mitochondrial plasmids ofvarious fungi and plants, and the polymerases from bacteriophages T4 andRB69 Family C polymerases are the primary bacterial chromosomereplicative enzymes. These are sometimes considered a subset of familyY, which contains the eukaryotic polymerase pol β, as well as othereukaryotic polymerases such as pol α, pol λ, pol μ, and terminaldeoxynucleotidyl transferase (TdT) Family D polymerases are all found inthe Euryarchaeota subdomain of Archaea and are thought to be replicativepolymerases. The family Y polymerases are called translesion synthesis(TLS) polymerases due to their ability to replicate through damaged DNA.They are also known as error-prone polymerases since they have a lowfidelity on undamaged templates. This family includes Pol η, Polζ, Pol ι(iota), Pol κ (kappa), and Rev1, and Pol IV and PolV from E coli.Finally, the reverse transcriptase family includes reversetranscriptases from retroviruses and eukaryotic polymerases, usuallyrestricted to telomerases. These polymerases use an RNA template tosynthesize the DNA strand, and are also known as RNA-dependent DNApolymerases.

In some embodiments, the modified polymerase includes one or more aminoacid mutations that are located outside the catalytic domains of thepolymerase. The catalytic domains of the A family DNA polymerases, Bfamily DNA polymerases and reverse transcriptases, as well as theRNA-dependent RNA polymerases are well known; all share a common overallstructure and catalytic mechanism. The catalytic domains of all thesepolymerases have a shape that has been compared to a right hand andconsists of “palm”, “thumb” and “finger” domains. The palm domaintypically contains the catalytic site for the phosphoryl transferreaction. The thumb is thought to play a role positioning the duplex DNAand in processivity and translocation. The fingers interact with theincoming nucleotide as well as the template base with which it ispaired. The palm domains are homologous in the A, B and RT families, butthe arrangements of the fingers and thumb are different. The thumbdomains of the different polymerase families do share common features,containing parallel or anti-parallel α-helices, with at least oneα-helix interacting with the minor groove of the primer-templatecomplex. The fingers domain also conserves an α-helix positioned at theblunt end of the primer-template complex. This helix contains highlyconserved side chains (the B motif).

Three conserved motifs, A, B, and C were originally identified for the Afamily polymerases. The A and C motifs were also found to be conservedin both the B family polymerases and the RT polymerases. (Delarue etal., Protein Engineering 3: 461-467 (1990)).

For the A family polymerases, the A motif comprises the consensussequence:

(SEQ ID NO: 10) DXSXXE. The B motif comprises the consensus sequence:(SEQ ID NO: 11) KXXXXXXYG The C motif comprises the consensus sequence(SEQ ID NO: 12) VHDE For the B family polymerases, the A motif comprises the consensus sequence: (SEQ ID NO: 13) DXXSLYPS.The B motif comprises the consensus sequence: (SEQ ID NO: 14) KXXXNSXYGThe C motif comprises the consensus sequence (SEQ ID NO: 15) YGDTDS

The residues in bold indicate invariant residues. In some embodiments,the modified polymerase includes amino acid mutations (e.g., amino acidsubstitutions, deletions, additions or chemical modifications located atany position other than the invariant residues.

The A and C motifs are part of the palm domain, and each contains astrictly conserved aspartic acid residue, which are involved in thecatalytic mechanism common to all the DNA polymerases. DNA synthesis ismediated by transfer of a phosphoryl group from the incoming nucleotideto the 3′ OH of the DNA, releasing a polyphosphate moiety and forming anew DNA phosphodiester bond. This reaction is catalyzed by a mechanisminvolving two metal ions, normally Mg²⁺, and the two conserved asparticacid residues.

The conserved glutamic acid residue in motif A of the A family DNApolymerases plays an important role in incorporation of the correctnucleotide, as does the corresponding conserved tyrosine in B familymembers (Minnick et al., Proc. Natl. Acad. Sci USA 99: 1194-1199 (2002);Parsell et al, Nucleic Acids Res. 35: 3076-3086 (2002). Mutations at theconserved Leu of motif A affect replication fidelity (Venkatesan et al.,J. Biol. Chem. 281: 4486-4494 (2006)).

The B motif contains conserved lysine, tyrosine and glycine residues.The B motif of E coli pol I has been shown to bind nucleotide substratesand contains a conserved tyrosine which has been shown to be in theactive site.

The B family polymerases contain six conserved motifs, of which regionsI and II correspond to the A and C motifs of the A family Region III isinvolved in nucleotide binding and is functionally homologous to motifB. Regions I, II and III converge at the center of the active site fromthe palm (I), the fingers (II), and base of the thumb (III) to produce acontiguous conserved surface. Within these regions, a set of highlyconserved residues form three chemically distinct clusters consisting ofexposed aromatic residues (Y416, Y567, and Y391), negatively chargedresidues (D621, D623, D411, D684, and E686), and a positively chargedcluster (K560, R482, and K486). These three clusters encompass theregion in which the primer terminus and the incoming nucleotide would beexpected to bind. (Wang et al, Cell 89: 1087-1099 (1997)).

The RT polymerases contain four conserved sequence motifs (Poch et al.,EMBO J. 12: 3867-3874 (1989)), with motifs A and C containing theconserved catalytic aspartates. The integrity of motif B is alsorequired for reverse transcriptase function.

(SEQ ID NO: 16) The consensus sequence for motif A is DXXXXF/Y (SEQ ID NO: 17) The consensus sequence for motif B is FXGXXXS/A (SEQ ID NO: 20) The consensus sequence for motif C is YXDD (SEQ ID NO: 21) The consensus sequence for motif D is GXXXXXXXK.

Mutations in the YXDD motif (motif C) (SEQ ID NO:20), the most highlyconserved of these motifs, can abolish polymerase activity and alter theprocessivity and fidelity. (Sharma et al., Antiviral Chemistry andChemotherapy 16: 169-182 (2005). In addition, the conserved lysineresidue in motif D, a loop that is unique to the RT polymerases, is aninvariant residue important for nucleotide binding (Canard et al., J.Biol. Chem. 274: 35768-35776 (1999).

In some embodiments, in addition to their polymerase domains, themodified polymerase can include one or more additional functionaldomains, including domains required for 3′→5′ (reverse) exonucleaseactivity that mediates proofreading of the newly synthesized DNA strand,or for 5′→3′ (forward) exonuclease activity that mediates nicktranslation during DNA repair. In some embodiments, the modifiedpolymerase has strand-displacing activity, and can catalyze nucleic acidsynthesis by polymerizing nucleotides into the 3′ end of a nick within adouble stranded nucleic acid template while simultaneously displacingthe nucleic acid located downstream of the nick.

The 3′ to 5′ exonuclease proofreading domains of both A and B family DNApolymerases contain three conserved motifs, called Exo I, Exo II and ExoIII, each of which contains an invariant aspartic acid residue essentialfor metal binding and exonuclease function. Alterations of theseconserved aspartic acid residues result in proteins which retainpolymerase activity, but are deficient in exonuclease activity. (Hall etal., J. Gen. Virol. 76: 2999-3008 (1995)). Conserved motifs in the 5′ to3′ exonuclease domains and amino acid alterations that affectexonuclease activity have also been identified (U.S. Pat. No.5,466,591).

Representative examples of A family enzymes are E. coli. Pol I, or theKlenow fragment of E coli. Pol I, Bst DNA polymerase, Taq DNApolymerase, T7 DNA polymerase and Tth DNA polymerase. A family enzymesalso include the Platinum Taq DNA polymerase series. Examples ofsuitable polymerases in this family include Phi-29 DNA polymerase, B103DNA polymerase, and the like.

It is generally thought that A family enzymes achieve high DNAelongation rates but have poor fidelity because of the lack of 3′-5′exonuclease activity, whereas B family enzymes have high fidelity owingto their 3′-5′ exonuclease activity but achieve low DNA elongationrates. It is possible to form mixed-type enzymes by mixing and it isgenerally thought that A family enzymes achieve high DNA elongationrates but have poor fidelity because of the lack of 3′-5′ exonucleaseactivity.

Unclassified types of enzymes include, for example, Tbr polymerase, Tflpolymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tmapolymerase, Tih polymerase, Tfi polymerase and the like. RT polymerasesinclude HIV reverse transcriptase, Moloney Murine Leukemia Virus (M-MLV)reverse transcriptase, Avian Myeloblastosis Virus (AMV) reversetranscriptase or Rous Sarcoma Virus (RSV) reverse transcriptase.Variants, modified products and derivatives thereof are also usable.

Of these enzymes, Taq, Platinum Taq, Tth, Tli, Pfu, Pfutubo, Pyrobest,Pwo and KOD, VENT, DEEPVENT, EX-Taq, LA-Taq, Therminator™, the Expandseries and Platinum Taq Hi-Fi are all commercially available. The otherenzymes can be readily isolated from specific bacteria by those ofordinary skill in the art.

One exemplary polymerase, E coli DNA polymerase I (“Pol I”) possessesthree enzymatic activities: a 5′ to 3′ DNA polymerase activity; a 3′ to5′ exonuclease activity that mediates proofreading; and a 5′ to 3′exonuclease activity mediating nick translation during DNA repair. TheKlenow fragment is a large protein fragment produced when E. coli Pol Iis proteolytically cleaved by subtilisin. It retains the polymerase andproofreading exonuclease activities, but lacks the 5′ to 3′ exonucleaseactivity. An exo-Klenow fragment which has been mutated to remove theproofreading exonuclease activity is also available. The structure ofthe Klenow fragment shows that highly conserved residues that interactwith DNA include N675, N678, K635, R631, E611, T609, R835, D827, S562and N579 (Beese et al, Science 260: 352-355 (1993)).

Arg682 in the Klenow fragment of E. coli DNA polymerase I (pol I) isimportant for the template-dependent nucleotide-binding function, andappears to maintain high processivity of the DNA polymerase. Pandey etal., European Journal of Biochemistry, 214:59-65 (1993).

In some embodiments, the modified polymerase is derived from the Bst DNApolymerase of Bacillus stearothermophilus, which is another A family DNApolymerase. The large fragment of the Bst DNA polymerase is equivalentto the Klenow fragment of E. coli Pol I, retaining the polymerase andproofreading exonuclease activities while lacking the 5′ to 3′exonuclease activity. Thus the term “Bst DNA polymerase” as used hereinmay refer to a full length protein or to a Bst large fragment.

In some embodiments, the modified polymerase is derived from Taq DNApolymerase, which is an A family DNA polymerase derived from thethermophilic bacterium Thermus aquaticus. It is best known for its usein the polymerase chain reaction. Taq polymerase lacks a proofreadingactivity, and thus has a relatively low replication fidelity. (Kim etal., Nature 376: 612-616 (2002).

In some embodiments, the modified polymerase is derived from the T7 DNApolymerase of bacteriophage T7, which is an A family DNA polymerase thatconsists of a 1:1 complex of the viral T7 gene 5 protein (80 k Da) andthe E. coli thioredoxin (12 k Da). It lacks a 5′→3′ exonuclease domain,but the 3′→5′ exonuclease activity is approximately 1000-fold greaterthan that of E coli Klenow fragment. The exonuclease activity appears tobe responsible for the high fidelity of this enzyme and prevents stranddisplacement synthesis. This polymerase is unique due to itsconsiderable processivity, or ability to stay on DNA for a greater thanaverage number of base pairs.

In some embodiments, the modified polymerase is derived from KOD DNApolymerase, which is a B family DNA polymerase derived from Thermococcuskodakaraensis. KOD polymerase is a thermostable DNA polymerase with highfidelity and processivity.

In some embodiments, the modified polymerase is derived from theTherminator™ DNA polymerase, which is also a B family DNA polymerase.Therminator™ is an A485L point mutation of the DNA polymerase fromThermococcus species 9oN-7. (Ichida et al., Nucleic Acids Res. 33:5214-5222 (2005). Therminator™ polymerase has an enhanced ability toincorporate modified substrates such as dideoxynucleotides,ribonucleotides, and acyclonucleotides.

In some embodiments, the modified polymerase is derived from a Phi29polymerase or a Phi29-type polymerase, for example a polymerase derivedfrom the bacteriophage B103. The Phi29 and B103 DNA polymerases are Bfamily polymerases from related bacteriophages. In addition to the A, Band C motifs, the Phi29 family of DNA polymerases contain an additionalconserved motif, KXY in region Y (Blanco et al., J. Biol. Chem. 268:16763-16770 (1993). Mutations to Phi29 and B103 polymerases that affectpolymerase activity and nucleotide binding affinity are described inU.S. Patent Publication No. 20110014612 and its priority documents U.S.Provisional Application Nos. 61/307,356; 61/299,917; 61/299,919;61/293,616; 61/293,618; 61/289,388; 61/263,974; 61/245,457; 61/242,771;61/184,770; and 61/164,324, herein incorporated by reference in theirentireties.

In some embodiments, the modified polymerase is derived from the reversetranscriptase from human immunodeficiency virus type 1 (HIV-1), which isa heterodimer consisting of one 66-kDa and one 51-kDa subunit. The p66subunit contains both a polymerase and an RNase H domain; proteolyticcleavage of p66 removes the RNase H domain to yield the p51 subunit.Wang et al., PNAS 91:7242-7246 (1994). The structure of the HIV-1reverse transcriptase shows multiple interactions between the 2′-OHgroups of the RNA template and the reverse transcriptase. ResiduesSer280 and Arg284 of helix I in the p66 thumb are involved in the RNA-RTinteractions, as well as residues Glu89 and Gln91 of the template gripin the p66 palm. The p51 subunit also plays a role in the interactionsbetween the RNA-DNA duplex and the RT, with residues Lys395, Glu396,Lys22 and Lys390 of the p51 subunit also interacting with the DNA:RNAduplex. (Kohlstaedt et al, Science 256: 1783-1790 (1992). Safarianos etal, The EMBO Journal 20:1449-1461 (2001)).

In some embodiments, the modified proteins of the disclosure can bederived from proteins, particularly nucleic acid binding proteins, whichcan be useful to include in nucleotide incorporation and/or nucleic acidsequencing applications. Many DNA polymerases interact with accessoryproteins such as the single-stranded DNA binding protein, the slidingclamp protein, and others. The DNA clamp proteins are multimericproteins that completely encircle the DNA and can significantly increasepolymerase processivity. The clamp proteins include the processivityfactor-sliding clamp for bacterial DNA polymerases II and III, gp45 forT4 DNA polymerase, UL44 for cytomegalovirus DNA polymerase, UL30 forherpes simplex virus I DNA polymerase, and proliferating cell nuclearantigen for eukaryotic DNA polymerases (Lee et al., J. Biol. Chem.295:1490-1499 (2010). Use of such modified proteins in ion-basednucleotide incorporation and sequencing reactions offers the sameadvantages as does use of modified polymerases, namely, reducedinterference with the ion-based sequencing signal, increased signal tonoise ratio, reduction of sequencing error rate, and increase in averageerror-free sequencing read length.

In some embodiments, the modified protein is derived from a singlestranded DNA binding protein. The single stranded DNA binding proteins(SSBs) are proteins that preferentially bind single stranded DNA (ssDNA)over double-stranded DNA in a nucleotide sequence independent mannerSSBs have been identified in virtually all known organisms, and appearto be important for DNA metabolism, including replication, recombinationand repair. Naturally occurring SSBs typically are comprised of two,three or four subunits, which may be the same or different. In general,naturally occurring SSB subunits contains at least one conserved DNAbinding domain, or “OB fold” (Philipova et al. (1996) Genes Dev.10:2222-2233; and Murzin (1993) EMBO J. 12:861-867), such that naturallyoccurring SSBs have four or more OB folds.

The best characterized SSB is that from E. coli, which exists as atetramer. (Raghunathan, Nature Structural & Molecular Biology 7, 648-652(2000). The E coli SSB (EcoSSB) monomer comprises an N-terminal domainrich in helices and sheets and a less structured C-terminal domain. TheN-terminal domain contains the OB fold, while the C-terminal domaincontains a highly conserved acidic region that weakens binding of EcoSSBto DNA, but may be involved in interactions with other proteins in vivo.The addition of single-stranded DNA-binding protein (SSB) in DNAsequencing reactions has been found to dramatically increase theresolution of sequencing runs. (Rapley (1994) Molecular Biotechnology 2:295-298.) SSBs are also used to increase the efficiency of PCR. (Kur etal., Acta Biochimica Polonica 52: 569-574 (2005). SSBs are availablecommercially and are used to improve the processivity and fidelity ofDNA polymerases (U.S. Patent Publication No. 20070059713).

In some embodiments, the modified proteins of the disclosure can bederived from DNA ligases. Ligases are essential components of DNAreplication, recombination, and repair systems found from viruses tohumans, catalyze the formation of a phosphodiester bond atsingle-stranded breaks on duplex DNA (Lehman, I. R., Science,186:790-797 (1974)). DNA ligases can be classified into two familiesbased on cofactor dependence. ATP-dependent ligases are found inbacteriophages (Dunn, et al., J Mol Biol., 148(4):303-330 (1981) andWeiss, et al., Proc Natl Acad Sci USA, 57(4):1021-1028 (1967)),Chlorella virus PBCV-1 (Ho, et al., J Virol, 71(3):1931-19374 (1997)),Vaccinia virus (Shuman, S., Biochemistry, 34(49):16138-161475 (1995)),Archea (Kletzin, A., Nucleic Acids Res, 20(20):5389-5396 (1992) andBult, et al., Science, 273(5278):1058-1073 (1996)), yeasts (Andaluz, etal., Yeast, 12(9):893-8988 (1996), Ramos, et al., Nucleic Acids Res,25(8); 1485-1492 (1997), Schar, et al., Genes Dev, 11(15):1912-1924(1997)), mammalian (Tomkinson, et al., Bioessays, 19(10):893-901 (1997),Tomkinson, et al., Mutat Res, 407(1):1-9 (1998), and Wang, et al., JBiol Chem, 269(50):31923-3192811 (1994)), and more recently eubacteria(Cheng, et al., Nucleic Acids Res, 25(7):1369-1374 (1997) and Deckert,et al., Nature, 392(6674):353-358 (1998)). NAD+ (i.e. nicotinamideadenine dinucleotide)-dependent ligases, however, are found exclusivelyin eubacteria. While some higher eucaryotic organisms may use multipleATP (i.e. adenosine triphosphate)-dependent ligases to fulfill diversebiological functions, some simple eubacteria genomes could host both anNAD+-dependent ligase and an ATP-dependent ligase (Deckert, et al.,Nature, 392(6674):353-358 (1998) and Fleischmann, et al., Science,269(5223):496-512 (1995)). The origin of the additional ATP-dependentligases in these genomes remains to be determined.

Although the ATP-dependent ligases and NAD+-dependent ligases sharelittle sequence homology, all the ligases investigated so far use thesame motif to form an adenylated enzyme intermediate (Tonikinson, etal., Bioessays, 19(10):893-901 (1997), Shuman, et al., Virology,211(1):73-83 (1995), and Luo, et al., Nucleic Acids Res,24(15):3079-3085 (1996)). Furthermore, they seem to be organized bysimilar domains and structural folds ((Doherty, et al., Nucleic AcidsRes, 24(12):2281-2287 (1996), Subramanya, et al., Cell, 85(4):607-615(1996), and Sekiguchi, et al., Nucleic Acids Res, 25(4):727-734 (1997)).

In some embodiments, the modified proteins of the disclosure can bederived from helicases, which are motor proteins that move directionallyalong a nucleic acid phosphodiester backbone, separating two annealednucleic acid strands (i.e., DNA, RNA, or an RNA-DNA hybrid) using energyderived from ATP hydrolysis. All helicases contain the classical WalkerA and B motifs, associated with ATP-binding and Mg2+-binding (reviewedin Caruthers and McKay. Curr. Opin. Struct. Biol. 12:123-133 (2002),Soultanas and Wigley. Trends Biochem. Sci. 26:47-54 (2001)). Helicaseshave been classified into several superfamilies (Gorbalenya and Koonin.Curr. Opin. Struct. Biol. 3:419-429 (1993)) according to the number ofhelicase signature motifs and differences in the consensus sequences formotifs. Superfamilies 1 and 2 have seven characteristic helicasesignature motifs and include helicases from archaea, eubacteria,eukaryotes and viruses, with helicases unwinding duplex DNA or RNA ineither 3′ to 5′ direction or 5′ to 3′ direction. Examples of superfamily1 helicases include the E. coli UvrD helicase, the T. tengcongensis UvrDhelicase, and the B subunit of RecBCD. Superfamily 3 has three motifsand superfamily 4 has five motifs. Examples of superfamily 4 helicasesinclude the T7 Gp4 helicase and DnaB helicases.

Helicases may be used in helicase dependent amplification (HDA) methodsfor in vitro DNA amplification. In contrast to PCR, which requiresthermocycling to separate the two DNA strands, HAD utilizes a DNAhelicase to generate single-stranded templates for primer hybridizationand subsequent primer extension by a DNA polymerase. Since the use of ahelicase eliminates the need for thermocycling, HDA can be performed ata single temperature for the entire process. (Vincent et al, EMBOReports 5: 795-800 (2004)).

Examples of naturally occurring DNA helicases, described by Kornberg andBaker in chapter 11 of their book, DNA Replication, W.H. Freeman andCompany (2nd ed. (1992)), include E. coli helicase I, II, III, & IV,Rep, DnaB, PriA, PcrA, T4 Gp41 helicase, T4 Dda helicase, T7 Gp4helicases, SV40 Large T antigen, yeast RAD. Additional helicases thatmay be useful in the disclosed sequencing methods include RecQ helicase(Harmon and Kowalczykowski, J. Biol. Chem. 276:232-243 (2001)),thermostable UvrD helicases from T. tengcongensis (U.S. Pat. No.7,829,284) and T. thermophilus (Collins and McCarthy, Extremophiles.7:35-41. (2003)), thermostable DnaB helicase from T. aquaticus (Kaplanand Steitz, J. Biol. Chem. 274:6889-6897 (1999)), and MCM helicase fromarchaeal and eukaryotic organisms ((Grainge et al., Nucleic Acids Res.31:4888-4898 (2003)).

In some embodiments, the modified proteins of the disclosure can bederived from a nuclease. Nucleases are enzymes that cleave thephosphodiester bonds between the nucleotide subunits of nucleic acids.Endonucleases cleave phosphodiester bonds within a polynucleotide chain,while exonucleases cleave phosphodiester bonds at the end of apolynucleotide chain.

During DNA synthesis the 3′ and 5′ exonucleases function to removeunwanted nucleotides from the DNA. Occasionally, a DNA polymerase willadd an incorrect nucleotide to the growing DNA polymer. A 3′ exonucleaseremoves nucleotides that have been incorrectly polymerized into DNAchains. These exonucleases are referred to as “proofreading”exonucleases.

In many cases the exonuclease activity is contained in the same proteinas the DNA polymerase activity. For example, the Escherichia coli DNApolymerase I is a single polypeptide with three separate domains, orregions of function. Each of these three domains contains an enzymaticactivity. The DNA polymerase activity is in one domain, and the twoother domains contain 3′ and 5′ exonuclease activities. The 3′exonuclease proofreads for the DNA polymerase, and the 5′ exonucleaseremoves unwanted nucleotides in advance of the DNA polymerase.

In some embodiments, the modified proteins of the disclosure can bederived from a topoisomerase. DNA topoisomerases are a specialized classof nucleases that bind to either single-stranded or double-stranded DNAand cut the phosphate backbone of the DNA. This intermediate breakallows the DNA to be untangled or unwound, and, at the end of theseprocesses, the DNA is reconnected again. Type I topoisomerases cut onestrand of a DNA double helix, while type II topoisomerases cut bothstrands of one DNA double helix, pass another unbroken DNA helix throughit, and then reanneal the cut strand. Type I topoisomerases include topoI, topo III and topo V. Type II topoisomerases include eukaryotic topoII, E. coli gyrase, E. coli topo IV, and topo VI. (Champoux JJ (2001)Annu. Rev. Biochem. 70: 369-413).

An example of a DNA topoisomerase that has been used in DNA sequencingreactions is Topoisomerase V from Methanopyrus kandleri, commerciallyavailable as Fidelase™. (U.S. Pat. No. 5,656,463). This thermostabletopoisomerase is used to enzymatically unlink and denature plasmid DNA,to help DNA polymerase pass through strong secondary structures and toprotect DNA from thermal decomposition.

In some embodiments, the disclosure also relates generally to methodsfor forming modified proteins having altered (e.g., increased ordecreased) buffering properties relative to the unmodified protein. Forexample, in some embodiments, the disclosure relates generally tomethods for forming modified polymerases having reduced bufferingcapacities for H⁺ ions within pH ranges of from about pH 4 to about pH10, typically from about pH 6 to about pH 9, even more typically fromabout pH 7 to about pH 9.

In some embodiments, the modified proteins of the disclosure includeproteins that have been modified to substitute, delete or chemicallymodify any chemical groups that have a pKa within the pH range ofinterest Amino acid side chains that are targeted for substitution,deletion or modification include those having a pKa within the range ofinterest, and which are likely to be on the surface of a protein.

In some embodiments, the selection of amino acid residues for mutationis based on analysis of the buffering properties, particularly the pKavalue, of the amino acids of the protein Amino acid residues having pKavalues that correspond to the pH range of desired reaction conditionsare likely to have stronger buffering capacities at the desired reactionconditions. For example, the modified proteins of the disclosure can beobtained by identifying amino acid residues within the protein thathave, or are predicted to have, pKa values falling outside typicalreaction conditions employed for the target protein-based assay, andreplacing or substituting one or more of the identified amino acidresidues with different amino acid residues having, or predicted tohave, pKa values outside the intended range of operation. Suchsubstitutions should reduce the buffering capacity of the overallprotein since the new amino acid residues will be expected to bufferonly weakly, if at all, under the reaction conditions that are typicallyemployed in applications using the protein.

In some embodiments, these general principles of amino acid selectionand mutation can be applied to produce modified nucleic acid bindingproteins (e.g., polymerases, ligases, helicases, SSBPs and the like),having reducing buffering capacities for H⁺ ions in primer extensionassays and nucleotide incorporation reactions (including nucleic acidsequencing reactions). In some embodiments, the pKa values of the aminoacid residues of a candidate protein can be predicted and evaluated toidentify all amino acid residues in the protein having pKa valuesfalling within the range of intended operating conditions for theprotein. Standard operating conditions for protein-based assaystypically correspond to physiological conditions and are well known inthe art. Standard operating conditions for protein assays, especiallyassays involving nucleic acid binding proteins and enzymes, typicallyinclude pH ranges of from about pH 4 to about pH 10, more typically fromabout pH 6 to about pH 9, even more typically from about pH to about pH9. In some embodiments, the pKa values of the amino acid residues of acandidate protein can be predicted and evaluated to identify all aminoacid residues in the protein having pKa values falling within the rangeof about pH 4 to about pH 10, more typically from about pH 6 to about pH9, even more typically from about pH 7 to about pH 9, therebyidentifying a first pool of candidate amino acid residues formodification.

In some embodiments, the amino acid residue to be modified is selectedbased not only on the pKa value but also on the degree of solventexposure of the amino acid residue. For example, the candidate aminoacid residues for modification can be selected based on their predictedpKa value (e.g., selecting all amino acid residues having, or predictedto have, a pKa falling within a desired pH range covering intendedoperating conditions) as well as their solvent exposures (e.g.,selecting amino acid residues having, or predicted to have, solventexposures of at least 30%, typically of at least about 40%, optionallyhaving solvent exposures of at least about 50%, 60%, 70%, 80%, or 90%).The solvent exposure of an amino acid in a protein measures the extentto which the amino acid is accessible to the solvent (usually water)surrounding the protein, a parameter that can also be variously referredto as surface exposure or solvation. The equivalent converse parameteris the degree to which an amino acid residue is buried (% buried) withinthe protein; an amino acid residue that is 20% buried is one that is 80%solvent exposed. The solvent accessibility (or conversely, the %burying) of an amino acid within a protein structure may be determinedby a variety of suitable methods, including by visual analysis of the athree-dimensional rendering of protein structure (e.g., crystalstructure), or by software analysis of the protein structure usingmethods known in the art (Lee and Richards, J. Mol. Biol. 55(3):379-400(1971); Shrake and Rupley, J. Mol. Biol. 79(2): 351-371 (1973);Connolly, J. Appl. Cryst. 16: 548-558 (1983)). A number of moleculargraphics programs are available which allow for the determination ofaccessible surface area of amino acids within a protein structure, suchas Rasmol, Charmm, AMBER, Swiss-PDBviewer, PropKa, and the like. Incases where the three-dimensional structure of the protein is not known,it may be modeled based upon sequence homology to a protein having aknown structure using molecular modeling methods known in the art.

In some embodiments, the first pool of candidate amino acids, which havebeen selected based on pKa values, can be further narrowed byidentifying all amino acid residues within the first pool having, orpredicted to have, at least a minimum threshold degree of surfaceexposure, also referred to as solvent exposure or solvation Amino acidresidues having, or predicted to have, degrees of solvation equal to orexceeding this threshold can be identified and selected to identify asecond pool of candidate amino acids for modification. In someembodiments, the second pool of candidate amino acids includes all aminoacid residues of the first pool having at least about 30% solventexposure, typically at least about 50% solvent exposure, even moretypically at least about 60% solvent exposure, even more typically atleast about 70% solvent exposure).

Methods for forming a modified protein can optionally include modifyingat least one of the amino acid residues in the candidate pool, therebyforming a modified protein having altered (e.g., increased or decreased)buffering capacities relative to the unmodified protein.

In some embodiments, one or more of the amino acid residues of the firstpool of candidate amino acids, or the second pool of candidate aminoacids, can optionally be substituted with a different amino acid residuehaving a pKa value falling outside the range of intended operation, forexample having pKa values that are outside the pH ranges of from aboutpH 4 to about pH 10, more typically outside the range of from about pH 6to about pH 9, even more typically outside the range of from about pH 7to about pH 9.

In some embodiments of the method, the comprises one or more amino acidsubstitutions that reduce the buffering capacity of said proteinrelative to the corresponding wild-type protein within the range ofabout pH 4 to about pH 10, wherein at least one of the one or more aminoacid substitutions includes a substitution of an amino acid residuehaving a pKa within the range of about 4.0 to about 10.0 with anotheramino acid residue. In some embodiments, the pKa of the amino acidresidue is a solution pKa of the amino acid residue. In someembodiments, particularly for internal amino acid residues lacking aprimary amine or free carboxylic group (e.g., non-terminal amino acidresidues), the pKa of the amino acid residue is approximated as the pKavalue of the side chain of the amino acid residue in solution. In otherembodiments, the pKa of the amino acid residue is a pKa of the aminoacid residue in the context of the corresponding wild-type protein(e.g., the “whole-protein pKa”).

In some embodiments, the modifying can include substituting at least oneamino acid residue having a pKa of between about 4.0 and about 10.0 withan amino acid residue having a pKa that is greater than about 10.0 orless than about 4.0. For example, the amino acid residue having a pKathat is greater than about 10.0 or less than about 4.0 can be selectedfrom the group consisting of: Arg, Asp, Gln, ly, Ile, Leu, Norleucine(Nle), Met, Phe, Ser, Thr, Trp, Val and N-terminal Formylmethionine(N-fMet).

In some embodiments, the modifying includes substituting at least oneamino acid residue having a pKa within the range of about 6.0 to about8.0 with another amino acid residue.

In some embodiments, the modifying includes substituting at least oneamino acid residue having a pKa of between about 6.0 and about 8.0 withan amino acid residue having a pKa that is greater than about 8.0 orless than about 6.0.

In some embodiments, the modifying includes substituting at least oneamino acid residue selected from the group consisting of: His, Glu, Asp,Tyr, and Lys, with any other different amino acid residue.

In some embodiments, the modifying includes substituting at least oneamino acid residue of the protein with an alanine residue.

In some embodiments, the modifying includes substituting at least oneamino acid residue that is at least 30% solvent exposed in thecorresponding wild-type protein with another amino acid residue.

In some embodiments of the method, the modifying includes introducing atleast one chemical amino acid modification that reduces the bufferingcapacity of said protein relative to the corresponding wild-type proteinwithin the range of about pH 4 to about pH 10. In some embodiments, theat least one chemical amino acid modification includes a chemicalmodification of the N-terminal amino acid. In some embodiments, the atleast one chemical amino acid modification includes a chemicalmodification of an amino acid residue including a primary amine groupwith an amine-reactive agent. In some embodiments, the amine-reactivereagent includes an acylating agent or an activated ester.

In some embodiments, the disclosure relates generally to methods forreducing the buffering capacity of a protein used in a DNA sequencing oramplification reaction, comprising making one or more conservative aminoacid substitutions in the protein sequence that substantially remove theprotein's buffering capacity within the range of pH 7 to pH 9.

Any suitable methods for designing and introducing modifications intoproteins may be used to design, engineer and synthesize the modifiedproteins of the disclosure. Methods of genetically engineering nucleicacid sequences encoding proteins to selectively introduce mutations,optionally in a site-specific fashion, are well known in the art ofrecombinant nucleic acid engineering, as are methods for cloning,expressing and purifying such sequences in vivo or in vitro. Someexemplary methods of cloning, expression and purification are describedin further detail herein.

In some embodiments, the protein can be modified by changing the pKavalues or ranges of selected amino acid residues. The pKa of acomposition (e.g., an amino acid) is a logarithmic measure of the aciddissociation constant Ka. An acid dissociation constant, Ka, (also knownas acidity constant, or acid-ionization constant) is a quantitativemeasure of the strength of an acid in solution. It is the equilibriumconstant for a chemical reaction known as dissociation in the context ofacid-base reactions. By modifying pKa values of amino acids in apolypeptide, the ability of the polypeptide to absorb hydrogen ions mayalter (e.g., increase or decrease), and/or its effect in ion detectionand buffering capacity.

The pKa values of amino acid side chains of a protein can play animportant role in defining the pH-dependent characteristics of aprotein. The pH-dependence of the activity displayed by enzymes and thepH-dependence of protein stability, for example, are properties that aretypically determined by the pKa values of amino acid side chains. ThepKa value of an amino acid residue may be the solution pKa value, a pKavalue based on the amino acid itself, but not on interaction with otheramino acids in the protein environment.

The pKa of the amino acid residues of a particular protein can beinferred or estimated or determined using any suitable method. Variousmethods for inferring the pKa value of a particular amino acid residueof a protein are known in the art. For example, the pKa values of aminoacid side chains can be inferred from the pKa values of model compoundsthat are similar to the side chains of amino acids. The pKas of theα-carboxylic acid, α-amino group, and titratable side chains for thefree amino acids in solution are well known in the art and available instandard tables, an example of which is provided in Table 1.

TABLE 1 Some Typical pKa Values For Free Amino Acids In Solution AminoAcid α-carboxylic acid α-amino Side chain Alanine 2.35 9.87 Arginine2.01 9.04 12.48 Asparagine 2.02 8.80 Aspartic Acid 2.10 9.82 3.86Cysteine 2.05 10.25 8.00 Glutamic Acid 2.10 9.47 4.07 Glutamine 2.179.13 Glycine 2.35 9.78 Histidine 1.77 9.18 6.10 Isoleucine 2.32 9.76Leucine 2.33 9.74 Lysine 2.18 8.95 10.53 Methionine 2.28 9.21Phenylalanine 2.58 9.24 Proline 2.00 10.60 Serine 2.21 9.15 Threonine2.09 9.10 Tryptophan 2.38 9.39 Tyrosine 2.20 9.11 10.07 Valine 2.29 9.72

In some embodiments, the solution pKa value may be a model pKa value.For example, the pKa values of an amino acid side chain in solution canbe inferred from the pKa values of model compounds (e.g., compounds thatare similar to the side chains of amino acids).

In some embodiments, the pKa value of an amino acid residue of a proteincan be estimated by simply approximating it to the value of its sidechain residue, as indicated in Table 1. In other words, the pKa value ofan amino acid residue (particularly an internal amino acid residue) canbe estimated as equal to the pKa value of its side chain, as indicatedin Table 1. An amino acid residue to be substituted, deleted or modifiedmay be selected based upon its solution pKa.

In some embodiments, the pKa value of an amino acid residue of a proteincan be estimated using available tools (e.g., software) that predict thepKa value of the amino acid residue in the context of the whole protein.The pKa value of a given amino acid residue of a folded protein(referred to herein as the “whole-protein pKa” of the amino acidresidue) can be different from the pKa of the free amino acid insolution (referred to herein as the “solution pKa” of the amino acidresidue). In a folded protein, an amino acid residue (including itstitratable amino acid side chain) may be buried within the interior ofthe protein and have limited or no exposure to solvent, and may alsointeract with and other titratable groups in the protein as well aspermanent charges (e.g. ions) and dipoles in the protein. All of theseeffects can alter the pKa value of the amino acid side chain from itssolution pKa. An amino acid residue to be substituted, deleted ormodified may also be selected based upon the whole-protein pKa of theamino acid residue in the wild type protein. The whole-protein pKa of anamino acid residue within a folded protein may be estimated by a varietyof techniques. Some methods are based on solutions to thePoisson-Boltzmann equation, often referred to as FDPB-based methods (for“finite difference Poisson-Boltzman). FDPB-based methods calculate thechange in the pKa value of an amino acid side chain when that side chainis moved from a hypothetical fully solvated state to its position in theprotein, using knowledge of the pKa values of amino acid side chains intheir fully solvated states combined with theoretical methods thatcalculate the effect of the protein interior on a pKa value. Publicallyavailable programs to calculate the estimated pKa values of amino acidside chains in a protein using FDPB methods include H++, available atthe H++ server at Virginia Polytechnic Institute (Gordon et al., NucleicAcids Research 33: W368-W371 (2005); Anandakrishnan and Onufriev,Journal of Computational Biology 15: 165-184 (2008)); Karlsberg+(Kieseritzky and Knapp, Proteins: Structure, Function, andBioinformatics 71:1335-1348 (2008)); Rabenstein and Knapp, BiophysicalJournal 80(3):1141-1150 (2001)); MCCE (Song and Gunner, J. Comp. Chemepub March 2009; Georgescu et al., Biophys J. 83, 1731-1748 (2002); andthe pKD webserver (Tynan-Connolly and Nielsen, Nucleic Acid Research 34:W48-W51 (2006); Tynan-Connolly and Nielsen, Protein Science 16: 239-249(2007)).

An alternative method is used by PropKa, a software program forcalculation of whole-protein pKa values available at the PROPKA WebInterface at the University of Copenhagen (Li et al., Proteins 61:704-721 (2005); Bas et al., Proteins 73: 765-783 (2008). The PropKaprogram for rapid prediction of pKa values is based upon a set ofempirical rules relating the protein structure to the pKa values ofionizable residues.

Molecular dynamics methods of calculating pKa values involvecomputationally measuring the free energy difference between theprotonated and deprotonated forms of the molecule. Molecular dynamics istypically a much more computationally expensive way to predict pKa'sthan using the Poisson-Boltzmann equation. In recent years, constant pHmolecular dynamics methods have been developed based on a microscopicdescription of the protein. (Wallace and Shen, Methods in Enzymology466: 455-475 (2009)).

The publicly available programs and webservers for pKa calculations ofamino acid side chains typically require a three-dimensional structureof the protein to be analyzed in PDB (Protein Data Bank) format. Thesestructures can be downloaded from sites such as PDBsum. (Laskowski,Nucleic Acids Res., 37, D355-D359 (2009)). The programs will return alisting of the calculated pKa values for the titratable amino acid sidechains of the protein, as well as the N-terminal and C-terminal groups.As an example, Tables 2 and 3 show the calculated pKas for amino acidswithin Bst DNA polymerase. Column 1 shows the amino acid residue, withnumbering based upon that of SEQ ID NO:1; column 2 shows the calculatedpKa of the side chain. The values shown in Table 2 were determined usingPropKa, while the values in Table 3 were determined using H++. Sincethese programs use different algorithms, they may return differentvalues for the pKas of individual amino acid resides. In some cases, itmay be useful to compare results from both programs as confirmation thata given amino acid residue has a pKa in a pH range of interest.

in some embodiments, the modifying can include substituting at least oneamino acid residue having an undesirable pKa (e.g., a pKa falling withinthe range of typical reaction conditions for the desired application)and likely to be on the protein surface (e.g., at least 30% solventexposed, more typically at least 50% solvent exposed) with an amino acidhaving a more desirable pKa (e.g., a pKa falling outside the range oftypical reaction conditions).

In some embodiments, the modifying can include substituting at least oneamino acid residue having a pKa of between about 4.0 and about 10.0 withan amino acid residue having a pKa that is greater than about 10.0 orless than about 4.0. In some embodiments, the amino acid residue havinga pKa that is greater than about 10.0 or less than about 4.0 is selectedfrom the group consisting of: Arg, Asp, Gln, ly, Ile, Leu, Norleucine(Nle), Met, Phe, Ser, Thr, Trp, Val and N-terminal Formylmethionine(N-fMet).

In some embodiments, the modifying can include substituting at least oneamino acid residue having a pKa within the range of about 6.0 to about8.0 with another amino acid residue. The pKa can optionally be thesolution pKa or the whole-protein pKa of the amino acid residue.

In some embodiments, the modifying can include substituting at least oneamino acid residue having a pKa of between about 6.0 and about 8.0 withan amino acid residue having a pKa that is greater than about 8.0 orless than about 6.0. In some embodiments, the amino acid to besubstituted is His, Glu, Asp, Tyr or Lys.

In some embodiments, the protein is a polymerase and the substitutingcan include replacement of any amino acid residue of the polymerase thatis not an invariant or conserved residue with another residue. Furtherdescriptions of amino acid residues that are conserved or invariantamongst polymerases are provided herein.

In some embodiments, the modifying can include introducing at least oneconservative substitution into the protein, in which at least oneproperty such as the size, shape or charge of the amino acid isconserved. A “conservative amino acid substitution” refers tosubstitution of a structurally and/or functionally similar amino acidthat may be made without not substantially altering the function of aprotein. An example of conservative substitution is the exchange of anamino acid in one of the following groups for another amino acid of thesame group (U.S. Pat. No. 5,767,063; Kyte and Doolittle, J. Mol. Biol.157: 105-132 (1982)):

(1) Hydrophobic: Norleucine, Ile, Val, Leu, Phe, Cys, Met;

(2) Neutral hydrophilic: Cys, Ser, Thr;

(3) Acidic: Asp; Glu;

(4) Basic: Asn, Gln, His, Lys, Arg;

(5) Residues that influence chain orientation: Gly, Pro;

(6) Aromatic: Trp; Tyr; Phe;

(7) Small amino acids: Gly, Ala, Ser.

For example, substitutions can be made by changing, e.g., Val to Leu;Ser to Thr; or Asp to Glu. Other substitutions can also be consideredconservative, depending on the environment of the particular amino acidand its role in the three-dimensional structure of the protein. Forexample, when it is desired to alter the pKa of an amino acid side chainwhile retaining the size and structure of the side chain, Glu may besubstituted by Gln, and Asp may be substituted by Asn.

In some embodiments, the amino acid residues selected for modification(e.g., replacement with another amino acid residue) are selected fromthe group consisting of: His, Glu, Asp, Cys, Lys and Tyr. In someembodiments, the amino acid residues selected for modification includeHis and Glu residues. These amino acid residues typically have pKavalues (even in the whole-protein context) of between about pH 6 toabout pH 8, and therefore possess high buffering capacities understandard physiological conditions and standard in vitro protein assayconditions. For example, His typically has a solution pKa (i.e., sidechain pKa) of about 6.1-6.5 under standard physiological conditions(e.g., pH conditions of from about pH 6.0 to about pH 9.0), while thatof Glutamine is about 4.1-4.5, and that of Cysteine is about 8.0.Replacement of these residues with amino acid residues that aretypically non-buffering under standard protein assay conditions can beadvantageous in applications in which it is desirable to achievelow-buffering conditions, e.g., ion-based reactions.

In some embodiments, the at least one conservative amino acidsubstitution is selected from the group consisting of His to Arg, Glu toGln, Asp to Asn, Lys to Arg, and Tyr to Phe.

In some embodiments, at least one of the one or more amino acidmodifications includes a substitution of an amino acid with an alanineresidue. Substitution or replacement of amino acid residues having highbuffering capacities with alanine residues can be advantageous inapplications in which it is desirable to reduce the buffering capacityof the protein. Introduction of alanine residues in lieu ofhigh-buffering residues is likely to reduce the overall bufferingcapacity of the protein because alanine residues typically are small,interfere minimally with protein structure, and typically have lowbuffering capacity under standard physiological conditions (or standardin vitro protein assay conditions) because they are usually uncharged atphysiological pHs. Alanine residues are therefore unlikely to possesssignificant buffering capacity under the operating conditions ofinterest.

In some embodiments, at least one of the one or more amino acidmodifications includes a deletion of an amino acid. In some embodiments,at least one of the one or more deleted amino acids includes an aminoacid residue having a pKa of between about 4.0 and about 10.0. In someembodiments, the amino acid residue having a pKa that is greater thanabout 10.0 or less than about 4.0 is selected from the group consistingof: Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr,Trp, Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more deleted amino acidsis an amino acid residue having a pKa within the range of about 6.0 toabout 8.0 with another amino acid residue. In some embodiments, thedeleted amino acid is His, Glu, Asp, Tyr or Lys.

An amino acid residue having an undesirable pKa may also be chemicallymodified so as to remove the buffering capacity of its titratable group.These modifications may include, for example, acylation or alkylation ofcysteine sulfhydryl groups, acylation of tyrosine, derivatization ofcarboxylate groups on aspartic acid or glutamic acid through the use ofamide bond forming agents or through active ester or reactive carbonylintermediates, and acylation or alkylation of amine groups of histidine,lysine arginine, or the N-terminal amine group, using conventionalreagents such as those disclosed in Hermanson (Bioconjugate Techniques,Academic Press, London, 2008). An N-terminal amine may be acylated usingN-succinimidyl-derived reagents, to attach acetate groups, polyethyleneglycol groups, biotins, or the like.

In some embodiments, after candidate protein is modified in the selectedfashion, it may be synthesized, and tested for altered bufferingcapacity. For example, testing for reduced or increased bufferingcapacity in a desired range, such as pH 7-9, may be accomplished byperforming a conventional pH titration on a known quantity of thecandidate protein and determining the characteristic of the titrationcurve in the desired pH range; namely, in the pH 7-9 range, smalladditions of hydroxide ion (or hydrogen ion) should correspond to largechanges in measured pH.

In some embodiments, the buffering capacity can be evaluated bycomparing the performance of a modified protein with a reference protein(e.g., a reference lacking at least one modification of the modifiedprotein, or the unmodified counterpart of the modified protein, or thewild-type version of the modified protein) in an ion-based sequencingreaction. In some embodiments, ion-based sequencing can be performedusing the Ion Torrent PGM™ sequencer (Life Technologies). In someembodiments, sequencing can be performed according to the user protocolssupplied with the PGM™ sequencer. Example 7 provides one exemplaryprotocol for ion-based sequencing using the Ion Torrent PGM™ sequencer,and also describes some of the sequencing performance metrics that canbe used to evaluate buffering capacity of the modified protein in asequencing reaction. In some embodiments, the number of 50Q17 reads canbe plotted against the number of keypass reads, and the results obtainedusing the modified and reference proteins can be compared to determinewhether the modified protein has altered buffering capacity relative tothe reference protein. In some embodiments, the number of 50Q17 readscan be plotted against the number of keypass reads, and the resultsobtained using the modified and reference proteins can be compared todetermine whether the modified protein has altered buffering capacityrelative to the reference protein. In some embodiments, the number ofcarryforwards can be plotted against the number of keypass reads, andthe results obtained using the modified and reference proteins can becompared to determine whether the modified protein has altered bufferingcapacity relative to the reference protein.

In some embodiments, measuring and comparing the performance of amodified protein with that of a reference protein can provide a usefultool for screening modified proteins for altered buffering capacities.For example, in some embodiments, the disclosure relates to a method forscreening modified proteins for altered buffering capacities,comprising: obtaining a candidate protein to be modified; introducingone or more modifications into the candidate protein, thereby producinga modified protein; using the modified protein in an ion-basedsequencing reaction and measuring the modified protein's performance inthe ion-based sequencing system according to a first parameter;measuring the performance of a reference protein in the ion-basedsequencing system according to the first parameter; and comparing theperformances of the modified and reference proteins in the ion-basedsequencing system according to the first parameter, thereby determiningwhether the modified protein has altered buffering capacity relative tothe reference protein. In some embodiments, the reference protein is aprotein lacking at least one modification of the modified protein. Insome embodiments, the reference protein is the unmodified counterpart ofthe modified protein. In some embodiments, the reference protein is thewild-type version of the modified protein. In some embodiments, thefirst parameter is the total number of 50Q17 reads obtained from asingle sequencing run. In some embodiments, the first parameter is thetotal number of 100Q17 reads obtained from a single sequencing run. Insome embodiments, the 50Q17 reads or the 100Q17 reads obtained using themodified and reference proteins can be plotted against the keypassreads, and the results can be compared.

In some embodiments, the modified protein is a polymerase and thereference protein is a wild-type version of the polymerase, or is anunmodified counterpart of the polymerase.

In some embodiments, the modified protein comprises one or more chemicalamino acid modifications that reduce the buffering capacity of theprotein relative to the corresponding wild-type protein within the rangeof about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, or about pH7 to about pH 9. In some embodiments, the one or more chemical aminoacid modifications includes a chemical modification of the N-terminalamino acid. In some embodiments, the one or more chemical amino acidmodifications includes a chemical modification of an amino acid residueincluding a primary amine group with an amine-reactive agent. In furtherembodiments, the amine-reactive reagent includes an acylating agent oran activated ester.

In some embodiments, at least one of the one or more amino acidsubstitutions, deletions or chemical modifications includes asubstitution of an amino acid residue that is at least 20%, at least25%, at least 30%, at least 35% or at least 40% solvent exposed in thecorresponding wild-type protein with another amino acid residue.

In some embodiments, the characteristic activity of the modified proteinis at least about 40%, 50%, 60%, 70%, 80% or 90% that of thecorresponding wild type protein. In order to retain the characteristicactivity of the modified protein, the sites of amino acid substitutions,deletions or modifications are chosen so as to avoid altering residuesknown to be important for protein function, such as residues that arehighly conserved across members of a protein family, or known catalyticsite residues.

In various embodiments, the modified protein has DNA- or RNA-bindingactivity. In various embodiments, the protein is selected from the groupconsisting of DNA polymerases, helicases, ligases, nucleases, singlestranded DNA binding proteins and polymerase accessory proteins. Thesemodified proteins may comprise any combination of amino acidsubstitutions, deletions or chemical modifications.

In some embodiments, the one or more amino acid substitutionssubstantially reduce the buffering capacity of said protein within therange of about pH 7 to pH 9. In some embodiments, at least one of theone or more amino acid substitution is a conservative amino acidsubstitution. In further embodiments, the at least one conservativeamino acid substitution is selected from the group consisting ofhistidine to arginine, glutamic acid to glutamine, aspartic acid toasparagine, lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, the protein is a DNA polymerase. In someembodiments, the disclosure relates generally to an isolated DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9. In some embodiments, the DNA polymerase is selected from the groupconsisting of an A family DNA polymerase; a B family DNA polymerase; amixed-type polymerase; an unclassified DNA polymerase and RT familypolymerase; and variants and derivatives thereof.

In some embodiments, the DNA polymerase is an A family DNA polymeraseselected from the group consisting of an A family DNA polymerase such asE. coli DNA polymerase, the Klenow fragment of E. coli DNA polymerase,Bst DNA polymerase, Taq DNA polymerase, Platinum Taq DNA polymeraseseries, T7 DNA polymerase, and Tth DNA polymerase. In some embodiments,the DNA polymerase is Bst DNA polymerase. In other embodiments, the DNApolymerase is E. coli DNA polymerase. In some embodiments, the DNApolymerase is the Klenow fragment of E. coli DNA polymerase. In someembodiments, the polymerase is Taq DNA polymerase. In some embodiments,the polymerase is T7 DNA polymerase.

In other embodiments, the DNA polymerase is a B family DNA polymeraseselected from the group consisting of Tli polymerase, Pfu polymerase,Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase,Sac polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, Pho polymerase, ES4 polymerase, VENT polymerase, DEEPVENTpolymerase, phage Phi29 polymerase, and phage B103 polymerase. In someembodiments, the polymerase is KOD polymerase. In some embodiments, thepolymerase is Therminator™ polymerase. In some embodiments, thepolymerase is phage Phi29 DNA polymerase. In some embodiments thepolymerase is phage B103 polymerase, including, for example, thevariants disclosed in U.S. Patent Publication No. 20110014612 and itspriority documents.

In other embodiments, the DNA polymerase is a mixed-type polymeraseselected from the group consisting of EX-Taq polymerase, LA-Taqpolymerase, Expand polymerase series, and Hi-Fi polymerase. In yet otherembodiments, the DNA polymerase is an unclassified DNA polymeraseselected from the group consisting of Tbr polymerase, Tfl polymerase,Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tihpolymerase, and Tfi polymerase.

In other embodiments, the DNA polymerase is an RT polymerase selectedfrom the group consisting of HIV reverse transcriptase, M-MLV reversetranscriptase and AMV reverse transcriptase. In some embodiments, thepolymerase is HIV reverse transcriptase or a fragment thereof having DNApolymerase activity.

In some embodiments, the DNA polymerase is a Bst DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Asused herein, a “Bst DNA polymerase” may refer to a full length proteinor to a Bst DNA polymerase large fragment which retains the polymeraseand proofreading domains while lacking the 5′ to 3′ exonuclease domain.

In some embodiments, the one or more conservative amino acidsubstitutions are of one or more amino acid residues shown in Table 2 orTable 3. Tables 2 and 3 show the calculated pKas for amino acids withinBst DNA polymerase. Column 1 shows the amino acid residue, withnumbering based upon that of SEQ ID NO:1; column 2 shows the calculatedpKa of the side chain; and column 3 indicates whether the amino acidresidue is on the surface of the protein (S) or is buried (B). Thevalues shown in Table 2 were determined using PropKa, while the valuesin Table 3 were determined using H++. In some embodiments, the one ormore conservative amino acid substitutions are of amino acid residueshaving a pKa of about 4.0 to about 10.0 as determined by bothalgorithms.

In some embodiments, the one or more conservative amino acidsubstitutions are selected from the group consisting of H46R, H273R,H281R, E446Q, H473R, H528R, H572R and Y477F, the numbering of amino acidresidues being in accordance with that of SEQ ID NO: 1.

In some embodiments, the Bst DNA polymerase comprises one or moreconservative amino acid substitutions, wherein the one or more aminoacid substitutions includes a substitution of alanine at position 2 withMet, Asn, Gln, Leu, Ile, Phe, or Trp, the numbering of amino acidresidues being in accordance with that of SEQ ID NO: 1.

In some embodiments, at least one of the one or more amino acidmodifications in the Bst DNA polymerase includes a substitution of anamino acid residue with an alanine residue. In some embodiments, the BstDNA polymerase comprises the substitution H528A.

In some embodiments, the disclosure relates generally to an isolated BstDNA polymerase comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a variantthereof having one or more conservative amino acid substitutions. Theinitial methionine residue of these amino acid sequences is not derivedfrom the native Bst polymerase, but is present to facilitate theproduction of the large fragment. Thus in some embodiments, the isolatedBst Polymerase comprises an amino acid sequence of any one of SEQ ID NO:2, SEQ ID NO: 3 and SEQ ID NO: 4, or any variant as described herein,wherein the protein lacks the N-terminal methionine residue.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 2. In some embodiments, the disclosure relatesgenerally to an isolated variant of a protein comprising the amino acidsequence shown in SEQ ID NO: 2, wherein the variant comprises an aminoacid sequence that is at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2.

In some embodiments, the disclosure relates generally to a Bst DNApolymerase comprising the amino acid sequence of SEQ ID NO: 3. In someembodiments, the disclosure relates generally to an isolated variant ofa protein comprising the amino acid sequence shown in SEQ ID NO: 3,wherein the variant comprises an amino acid sequence that is at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identical to SEQ ID NO: 3.

In some embodiments, the disclosure relates generally to a Bst DNApolymerase comprising the amino acid sequence of SEQ ID NO: 4. In otherembodiments, the disclosure relates generally to an isolated variant ofa protein comprising the amino acid sequence shown in SEQ ID NO: 4,wherein the variant comprises an amino acid sequence that is at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identical to SEQ ID NO: 4.

In some embodiments, the DNA polymerase is a Therminator™ DNA polymerasecomprising one or more amino acid substitutions, deletions or mutationsas described above. In some embodiments, the Therminator™ DNA polymerasecomprises one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine.

In some embodiments, the one or more conservative amino acidsubstitutions are of one or more amino acid residues shown in Table 5.Table 5 shows the calculated pKas for amino acids within Therminator™DNA polymerase as determined using PropKa. Column 1 shows the amino acidresidue, with numbering based upon that of SEQ ID NO: 5; while column 2shows the calculated pKa of the side chain.

In some embodiments, the DNA polymerase is a KOD DNA polymerasecomprising one or more amino acid substitutions, deletions or mutationsas described above. In some embodiments, the KOD DNA polymerasecomprises one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 6. Table 6 showsthe calculated pKas for amino acids within KOD DNA polymerase asdetermined using PropKa. Column 1 shows the amino acid residue, withnumbering based upon that of SEQ ID NO: 6; while column 2 shows thecalculated pKa of the side chain. Optionally, the KOD polymeraseincludes one or more mutations reducing an exonuclease activity (forexample, a 3′→5′exonuclease activity) of the protein.

In some embodiments, the DNA polymerase is a B103 DNA polymerasecomprising one or more amino acid substitutions, deletions or mutationsas described above. A B103 DNA polymerase may include any of the variantB103 polymerases, or biologically active fragments thereof, as disclosedin U.S. Patent Publication No. 20110014612, which is incorporated byreference herein.

In some embodiments, the B103 polymerase has the amino acid sequence ofSEQ ID NO: 7, further comprising amino acid substitutions at positions383 and 384, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 7. In some embodiments, the polymerase comprisesthe amino acid substitutions F383L and D384N, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the B103 polymerase has the amino acid sequence ofSEQ ID NO: 8.

(SEQ ID NO: 8)   1mprkmfscdf etttklddcr vwaygymeig nldnykigns ldefmqwvme iqadlyfhnl  61kfdgafivnw lehhgfkwsn eglpntynti iskmgqwymi dicfgykgkr klhtviydsl 121kklpfpvkki akdfqlpllk gdidyhaerp vgheitpeey eyikndieii araldiqfkg 181gldrmtagsd slkgfkdils tkkfnkvfpk lslpmdkeir rayrggftwl ndkykekeig 241egmvfdvnsl ypsqmysrpl pygapivfqg kyekdegypl yiqrirfefe lkegyiptiq 301ikknpffkgn eylknsgaep velyltnvdl eliqehyemy nveyidgfkf rektglfkef 361idkwtyvkth ekgakkqlak lmfdslygkf asnpdvtgkv pylkedgslg frvgdeeykd 421pvytpmgvfi tawarfttit aaqacydrii ycdtdsihlt gtevpeiikd ivdpkklgyw 481ahestfkrak ylrqktyiqd iyakevdgkl iecspdeatt tkfsvkcagm tdtikkkvtf 541dnfrvgfsst gkpkpvqvng gvvlvdsvft ik

In some embodiments, the B103 polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 8, or any biologically activefragment thereof. Typically, a polymerase having the amino acid sequenceof SEQ ID NO: 8 will exhibit increased polymerase activity (e.g., primerextension activity) relative to the polymerase having the amino acidsequence of SEQ ID NO: 7.

In some embodiments, the B103-type polymerase comprises one or moremodifications resulting in altered exonuclease activity (for example 3′to 5′ exonuclease activity) as compared to a reference polymerase. Insome embodiments, the modification comprises an amino acid substitution,for example the amino acid substitution D166A. In some embodiments, themodified B103-type polymerase lacks 3′ to 5′ exonuclease activity, orlacks 5′ to 3′ exonuclease activity, or both. Mutations that reduce oreliminate 3′ to 5′ exonuclease activity have been described, forexample, in Phi-29 polymerase at various residues. See, e.g., de Vega etal., EMBO J., 15(5):1182-1192 (1996); Soengas et al., EMBO J.,11(11):4227-4237 (1992); Blanco et al., U.S. Pat. Nos. 5,001,050,5,198,543 and 5,576,204.

In some embodiments, the B103 polymerase comprises an amino acidsequence that is at least 70%, 75%, 85%, 90%, 95% or 99% identical toany one of the amino acid sequences SEQ ID NO: 7, SEQ ID NO: 8 and SEQID NO: 9, or to any biologically active fragment thereof, and furthercomprises one or more amino acid substitutions, additions or deletionsat one or more positions selected from the group consisting of: 2, 9,11, 12, 58, 59, 63, 162, 166, 377 and 385, wherein the numbering isrelative to SEQ ID NO: 7. In some embodiments, this polymerase canexhibit reduced exonuclease activity relative to an unmodifiedcounterpart.

In some embodiments, the B103 polymerase comprises an amino acidsequence that is at least 70%, 75%, 85%, 90%, 95% or 99% identical toany one of the amino acid sequences SEQ ID NO: 7 or SEQ ID NO: 8 or toany biologically active fragment thereof, and further comprises theamino acid mutation D166A, wherein the numbering is relative to SEQ IDNO: 7. In some embodiments, this modified polymerase can exhibit reduced3′ to 5′ exonuclease activity relative to a reference polymerase havingthe amino acid sequence of SEQ ID NO: 7.

In some embodiments, the B103 DNA polymerase comprises an amino acidsequence that is at least 70%, 75%, 85%, 90%, 95% or 99% identical toany one of the amino acid sequences SEQ ID NO: 7, SEQ ID NO: 8 and SEQID NO: 9, or to any biologically active fragment thereof, and furthercomprises one or more amino acid substitutions selected from the groupconsisting of: D9A, E11A, E11I, T12I. H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to SEQ ID NO:7. In some embodiments, this modified polymerase can exhibit reducedexonuclease activity relative to an unmodified counterpart.

In some embodiments, the B103 DNA polymerase comprises an amino acidsequence that is at least 70%, 75%, 85%, 90%, 95% or 99% identical toany one of the amino acid sequences SEQ ID NO: 7, SEQ ID NO: 8 and SEQID NO: 9 or to any biologically active fragment thereof, and furthercomprises one or more amino acid substitutions selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,Q377A and S385G, wherein the numbering is relative to SEQ ID NO: 7.Typically, this modified polymerase can exhibit reduced 3′ to 5′exonuclease activity relative to an unmodified counterpart.

In some embodiments, the B103 DNA polymerase comprises an amino acidsequence that is at least 85%, 90%, 95% or 99% identical to the aminoacid sequence of SEQ ID NO: 7 and further comprises an amino acidsubstitution wherein the amino acid residue at position 9 is replacedwith an alanine (“A”) residue, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 7.

In some embodiments, the B103 DNA polymerase comprises an amino acidsequence that is at least 85%, 90%, 95% or 99% identical to the aminoacid sequence of SEQ ID NO: 7 and further comprises one or more of theamino acid substitutions D9A, E11A, T12I, H58R, N59D, D63A, D166A,Q377A, S385G, or any combination thereof, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 7. In someembodiments, the B103 DNA polymerase comprises any one, two, three,four, five or all of these mutations. In some embodiments, the B103 DNApolymerase comprises the amino acid substitutions D9A and D63A. In someembodiments, the B103 DNA polymerase comprises the amino acidsubstitutions N59D and T12I. Typically, this polymerase will exhibitreduced 3′ to 5′ exonuclease activity relative to an unmodifiedcounterpart.

In some embodiments, the B103 DNA polymerase comprises an amino acidsequence that is at least 85%, 90%, 95% or 99% identical to the aminoacid sequence of SEQ ID NO: 8, and comprises any one, two, three or moreof the mutations described herein.

Optionally, the modification(s) reducing 3′ to 5′ exonuclease activitycan be combined with additional modification(s) that increase polymeraseactivity. Modifications increasing polymerase activity include, forexample, the amino acid substitutions F383L and D384N.

In some embodiments, the B103 DNA polymerase comprises the amino acidsequence of SEQ ID NO: 7 and further comprises the amino acidsubstitution D166A, which reduces the 3′ to 5′ exonuclease activity, incombination with the amino acid substitutions F383L and D384N, whichincrease the polymerase activity of the substituted polymerase, relativeto the unmodified protein having the amino acid sequence of SEQ ID NO:8. The amino acid sequence of this triple mutant polymerase is the aminoacid sequence of SEQ ID NO: 9, below:

(SEQ ID NO: 9)   1mprkmfscdf etttklddcr vwaygymeig nldnykigns ldefmqwvme iqadlyfhnl  61kfdgafivnw lehhgfkwsn eglpntynti iskmgqwymi dicfgykgkr klhtviydsl 121kklpfpvkki akdfqlpllk gdidyhaerp vgheitpeey eyiknaieii araldiqfkg 181gldrmtagsd slkgfkdils tkkfnkvfpk lslpmdkeir rayrggftwl ndkykekeig 241egmvfdvnsl ypsqmysrpl pygapivfqg kyekdegypl yiqrirfefe lkegyiptiq 301ikknpffkgn eylknsgaep velyltnvdl eliqehyemy nveyidgfkf rektglfkef 361idkwtyvkth ekgakkqlak lmlnslygkf asnpdvtgkv pylkedgslg frvgdeeykd 421pvytpmgvfi tawarfttit aaqacydrii ycdtdsihlt gtevpeiikd ivdpkklgyw 481ahestfkrak ylrqktyiqd iyakevdgkl iecspdeatt tkfsvkcagm tdtikkkvtf 541dnfrvgfsst gkpkpvqvng gvvlvdsvft ik

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%identical to the amino acid sequence of SEQ ID NO: 9, or anybiologically active fragment thereof. Typically, the modified polymeraseof SEQ ID NO: 9 will exhibit reduced exonuclease activity relative to areference polymerase comprising the amino acid sequence of SEQ ID NO: 7or SEQ ID NO: 8.

In some embodiments, the disclosure relates generally to a B103 DNApolymerase, including any of the variants described above, wherein theB103 DNA polymerase comprises one or more conservative amino acidsubstitutions that substantially reduce its buffering capacity relativeto the corresponding unmodified protein within the range of about pH 7to about pH 9, wherein the one or more conservative amino acidsubstitutions are selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table7. Table 7 shows the calculated pKas for amino acids within Therminator™DNA polymerase as determined using PropKa. Column 1 shows the amino acidresidue, with numbering based upon that of SEQ ID NO: 7; while column 2shows the calculated pKa of the side chain.

In some embodiments, the modified polymerase retains polymeraseactivity. In various embodiments, the polymerase activity of a modifiedDNA polymerase is at least about 40%, 50%, 60%, 70%, 80% or 90% that ofthe corresponding wild type protein. As used herein, the term“polymerase activity” and its variants, when used in reference to agiven polymerase, comprises any in vivo or in vitro enzymatic activitycharacteristic of a given polymerase that relates to catalyzing thepolymerization of nucleotides into a nucleic acid strand, e.g.,nucleotide incorporation activity (typically measured as primerextension activity in a primer extension assay), and the like.Typically, but not necessarily such nucleotide polymerization occurs ina template-dependent fashion. In addition to such polymerase activity,the polymerase can typically possess other enzymatic activities, forexample, 3′ to 5′ exonuclease activity, 5′ to 3′ exonuclease activity,and the like.

In some embodiments, the amount of nucleotide incorporation activity (orprimer extension activity) exhibited by the polymerase can be quantifiedas the total number of nucleotides incorporated (as measured by, e.g.,radiometric or other suitable assay) by a unit amount of polymerase (inmoles) per unit time (seconds) under a particular set of reactionconditions. The nucleotide incorporation activity (or primer extensionactivity) can be measured using any suitable assay that provides aquantitative indication of the amount of extension product obtainedusing defined reaction conditions comprising a known concentration ofpolymerase. Regardless of which assay is used, differences in nucleotideincorporation activity (or primer extension activity) between twosamples, when obtained using identical reaction conditions, can beevaluated by simply comparing levels of observed nucleotideincorporation activity (or primer extension activity) obtained from eachsample. Optionally, the observed nucleotide incorporation activity (orprimer extension activity) can normalized for amount of polymerase bydividing the amount of incorporated radioactivity by the polymeraseconcentration in the reaction mixture, to allow comparison betweenreactions containing different polymerase concentrations.

In one exemplary embodiment, the nucleotide incorporation activity (orprimer extension activity) of a polymerase can be measured using aradiometric assay that measures incorporation of a radioactively labelednucleotide into acid-insoluble material in a polymerase reaction. Theamount of incorporated radioactivity indicates the total number ofnucleotides incorporated. See, e.g., Wu et al., Gene Biotechnology, 2ndEd., CRC Press; Sambrook, J., Fritsch, E F, and Maniatis, T. (1989)Molecular Cloning A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

In another exemplary embodiment, levels of nucleotide incorporationactivity (or primer extension activity) in a sample can be measured bymonitoring the fluorescence intensity change over time during extensionof a fluorescein-labeled hairpin oligonucleotide.

In another exemplary embodiment, the nucleotide incorporation activity(or primer extension activity) of a given polymerase can be quantifiedby quantifying the amount of pyrophosphate liberated after performingnucleotide incorporation (e.g., primer extension) under standardtolerance assay conditions for 5 minutes.

Various examples of such assays for polymerase activity can be found,for example, in U.S. application Ser. No. 12/748,359, now published asU.S. Publication No. 20110014612.

In order to retain the characteristic activity of the modified protein,the sites of amino acid substitutions, deletions or chemicalmodifications are chosen so as to avoid altering residues known to beimportant for protein function, such as residues that are highlyconserved across members of the particular family of DNA polymerases towhich the modified protein belongs, or known catalytic site residuesinvolved in polymerase activity.

In some embodiments, the modified polymerase that retains polymeraseactivity is a B103 DNA polymerase Amino acid residues that affect thepolymerase or exonuclease activity of the B103 DNA polymerase have beendescribed in detail above.

In some embodiments, the modified polymerase that retains polymeraseactivity is a Bst DNA polymerase. The amino acid sequence of the largefragment of the Bst DNA polymerase protein is shown in SEQ ID NO: 1:

MAKMAFTLAD RVTEEMLADK AALVVEVVEE NYHDAPIVGI AVVNEHGRFF LRPETALADP  60QFVAWLGDET KKKSMFDSKR AAVALKWKGI ELCGVSFDLL LAAYLLDPAQ GVDDVAAAAK 120MKQYEAVRPD EAVYGKGAKR AVPDEPVLAE HLVRKAAAIW ELERPFLDEL RRNEQDRLLV 180ELEQPLSSIL AEMEFAGVKV DTKRLEQMGK ELAEQLGTVE QRIYELAGQE FNINSPKQLG 240VILFEKLQLP VLKKTKTGYS TSADVLEKLA PYHEIVENIL HYRQLGKLQS TYIEGLLKVV 300RPDTKKVHTI FNQALTQTGR LSSTEPNLQN IPIRLEEGRK IRQAFVPSES DWLIFAA DYS 360QIELRVLAHI AEDDNLMEAF RRDLDIHTKT AMDIFQVSED EVTPNMRRQA  KAVNFGIVYG 420ISDYGLAQNL NISRKEAAEF IERYFESFPG VKRYMENIVQ EAKQKGYVTT LLHRRRYLPD 480ITSRNFNVRS FAERMAMNTP IQGSAADIIK KAMIDLNARL KEERLQAHLL LQVHDELILE 540APKEEMERLC RLVPEVMEQA VTLRVPLKVD YHYGSTWYDA K

SEQ ID NO: 1 contains the DNA polymerase motifs A, B, and C (Delahue,supra) at residues 358-363, 411-420 and 533-536, respectively, as shownin FIG. 1. The motifs are underlined and highlighted, and the invariantresidues within each motif are indicated in bold. Thus in order toretain the polymerase activity of a modified Bst polymerase, anysubstitutions, deletions or chemical modifications will be made to aminoacid residues that are not highly conserved within motifs A, B or C,such as the invariant aspartic acid residues D358 and D535 required forpolymerase activity.

In some embodiments, the modified Bst DNA polymerase retainsproofreading exonuclease activity. In various embodiments, theproofreading exonuclease activity of a modified Bst DNA polymerase is atleast about 40%, 50%, 60%, 70%, 80% or 90% that of the correspondingwild type protein. In order to retain the proofreading exonucleaseactivity of a modified Bst DNA polymerase, the skilled artisan willunderstand that any substitutions, deletions or chemical modificationsshould be made to amino acid residues that are not highly conservedwithin the Exo I, Exo II and Exo III motifs.

In some embodiments, the disclosure relates generally to an isolatedprotein comprising one or more amino acid substitutions, deletions, orchemical modifications that reduce the buffering capacity of saidprotein relative to the corresponding wild-type protein within the rangeof about pH 4 to about pH 10, wherein the one or more amino acidsubstitutions, deletions or chemical modifications substantially reducethe average number of sequencing errors obtained from an ion-basedsequencing reaction using the modified protein, relative to the numberof sequencing errors obtained from an ion-based sequencing reactionusing the corresponding wild-type (unmodified) protein.

The sequencing error rate will in part depend upon the fidelity of thepolymerase used in the sequencing reaction. The fidelity of a polymerasedepends in part upon the tendency of the polymerase to incorporateincorrect nucleotides, as well as the presence of an integral 3′ to 5′exonuclease activity, which can increase fidelity by excisingmisincorporated nucleotides. It is known that a conserved residue inmotif A of DNA polymerases (E for family A members and Y for family Bmembers), as well as residues which interact with this conservedresidue, play an important role in incorporation of the correctnucleotide (Parsell, supra; Minnick, supra). Conserved motifs involvedin 3′ to 5′ exonuclease activity are also known, as discussed above. Itis also known in the art that some polymerases have improved fidelityrelative to others. Residues that may play a role in either nucleotideincorporation or 3′ to 5′ exonuclease activity may be determined by areview of the relevant motifs in a polymerase, or by comparison topolymerases having improved fidelity. Thus in some embodiments, thedisclosure relates generally to a modified DNA polymerase, wherein oneor more of the modifications to the polymerase affect either nucleotideincorporation or 3′ to 5′ exonuclease activity.

In some embodiments, the disclosure relates generally to an isolatedprotein comprising one or more amino acid substitutions, deletions orchemical modifications that reduce the buffering capacity of saidprotein relative to the corresponding wild-type protein within the rangeof about pH 4 to about pH 10, wherein the one or more amino acidsubstitutions, deletions or chemical modifications increase the averagelength of sequencing reads having at least 90% accuracy obtained from anion-based sequencing reaction using the modified protein, relative toaverage length of sequencing reads having 90% accuracy obtained from anion-based sequencing reaction using the corresponding wild-type(unmodified) protein.

The length of sequencing reads depends in part upon the processivity ofthe polymerase used in the sequencing reaction. The processivity of apolymerase is a measure of the average number of nucleotides added bythe polymerase per association/disassociation with the template.

It is known in the art that some polymerases have improved processivityrelative to others. Residues that may play a role in processivity may bedetermined by comparison to polymerases having improved processivity.Thus in some embodiments, the disclosure relates generally to a modifiedDNA polymerase, wherein one or more of the modifications to thepolymerase affect processivity.

It is also known in the art that accessory proteins, including, forexample, sliding clamp proteins or single stranded DNA binding proteins(SSBs), can improve the processivity of a DNA polymerase. SSBs are alsoused in the art to improve fidelity of DNA polymerases. Thus in someembodiments, the disclosure relates generally to a modified accessoryprotein or SSB, wherein the modified protein provides increasedimprovements to the processivity and/or fidelity of a polymerase used ina sequencing reaction, as compared to an unmodified accessory protein orSSB.

In some embodiments, the isolated protein comprising one or more aminoacid substitutions that reduce the buffering capacity of said proteinrelative to the corresponding wild-type protein within the range ofabout pH 4 to about pH 10 is a single stranded DNA binding protein(SSB).

In some embodiments, the SSB comprises one or more conservative aminoacid substitutions that substantially reduce its buffering capacitywithin the range of pH 7 to pH 9. In further embodiments, the one ormore conservative amino acid substitutions are selected from the groupconsisting of histidine to arginine, glutamic acid to glutamine,aspartic acid to asparagine, lysine to arginine, and tyrosine tophenylalanine. In some embodiments, the SSB is E. coli SSB. In furtherembodiments, the one or more conservative amino acid substitutions areof one or more amino acid residues shown in Table 4. Tables 2 and 3 showthe calculated pKas for amino acids within E. coli SSB. Column 1 showsthe amino acid residue, with numbering based upon that of SEQ ID NO:1;column 2 shows the calculated pKa of the side chain. In someembodiments, the SSB comprises the amino acid substitution K7R.

In some embodiments, any of the isolated proteins described hereinfurther include an affinity tag, a label, a chemical moiety, aradionuclide, an enzyme, a fluorescent marker, a chemiluminescentmarker, or a combination thereof.

In some embodiments, the isolated protein is coupled to a polymer, asupport, or a sensor. Polymers may include, for example, polyethyleneglycol, PEA, a dextran, an acrylamide, or a cellulose (e.g., methylcellulose). Supports may include, for example, beads or other solidsurfaces such as the surface of a reaction chamber, microwell or sensor.Sensors may include, for example, a FET. In some embodiments, the sensoris a chemFET. In some embodiments, the sensor is an ISFET.

In some embodiments, the disclosure relates generally to a method forincorporating at least one nucleotide into a primer, comprising:contacting a nucleic acid duplex including a template nucleic acid and aprimer with a modified polymerase according to the disclosure in thepresence of one or more nucleotides, and incorporating at least onenucleotide into the primer in a template-dependent fashion using thepolymerase. The modified polymerase may be any of the polymerases havingany of the modifications disclosed above, including one or more aminoacid substitutions, deletions and chemical modifications.

In some embodiments of the method, the polymerase comprises one or moreamino acid substitutions that reduce the buffering capacity of saidprotein relative to the corresponding wild-type protein within the rangeof about pH 4 to about pH 10. In some embodiments, the one or more aminoacid substitutions reduce the buffering capacity of the polymeraserelative to the corresponding wild-type polymerase within the range ofabout pH 7 to about pH 9, or of about pH 6 to about pH 8.

In some embodiments, the one or more amino acid substitutionssubstantially reduce the buffering capacity of the polymerase within therange of about pH 7 to pH 9. In embodiments, at least one of the one ormore amino acid substitution is a conservative amino acid substitution.In further embodiments, the at least one conservative amino acidsubstitution is selected from the group consisting of histidine toarginine, glutamic acid to glutamine, aspartic acid to asparagine,lysine to arginine, and tyrosine to phenylalanine.

In various embodiments of the method, the DNA polymerase is selectedfrom the group consisting of an A family DNA polymerase; a B family DNApolymerase; a mixed-type polymerase; an unclassified DNA polymerase andRT family polymerase; and variants and derivatives thereof.

In some embodiments, the DNA polymerase is an A family DNA polymeraseselected from the group consisting of a Pol I-type DNA polymerase suchas E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, Bst DNA polymerase, Taq DNA polymerase, Platinum Taq DNApolymerase series, T7 DNA polymerase, and Tth DNA polymerase. In someembodiments, the DNA polymerase is Bst DNA polymerase. In otherembodiments, the DNA polymerase is E. coli DNA polymerase. In someembodiments, the DNA polymerase is the Klenow fragment of E. coli DNApolymerase. In some embodiments, the polymerase is Taq DNA polymerase.In some embodiments, the polymerase is T7 DNA polymerase.

In other embodiments, the DNA polymerase is a B family DNA polymeraseselected from the group consisting of: Bst polymerase, Tli polymerase,Pfu polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase,KOD polymerase, Sac polymerase, Sso polymerase, Poc polymerase, Pabpolymerase, Mth polymerase, Pho polymerase, ES4 polymerase, VENTpolymerase, DEEPVENT polymerase, Therminator™ polymerase, phage Phi29polymerase, and phage B103 polymerase. In some embodiments, thepolymerase is KOD polymerase. In some embodiments, the polymerase isTherminator™ polymerase. In some embodiments, the polymerase is phagePhi29 DNA polymerase. In some embodiments the polymerase is derived fromphage B103 polymerase, including, for example, the variants disclosed inU.S. Patent Publication No. 20110014612 which is incorporated byreference herein.

In other embodiments, the DNA polymerase is a mixed-type polymeraseselected from the group consisting of EX-Taq polymerase, LA-Taqpolymerase, Expand polymerase series, and Hi-Fi polymerase. In yet otherembodiments, the DNA polymerase is an unclassified DNA polymeraseselected from the group consisting of Tbr polymerase, Tfl polymerase,Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tihpolymerase, and Tfi polymerase.

In some embodiments, the DNA polymerase is an RT polymerase selectedfrom the group consisting of HIV reverse transcriptase, M-MLV reversetranscriptase and AMV reverse transcriptase. In some embodiments, thepolymerase is HIV reverse transcriptase or a fragment thereof having DNApolymerase activity.

In some embodiments of the method, the DNA polymerase is a Bst DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 2 or Table 3. Insome embodiments, the one or more conservative amino acid substitutionsare selected from the group consisting of H46R, H273R, H281R, E446Q,H473R, H528R, H572R and Y477F, the numbering of amino acid residuesbeing in accordance with that of SEQ ID NO: 1.

In some embodiments, the Bst DNA polymerase comprises one or moreconservative amino acid substitutions, wherein the one or more aminoacid substitutions includes a substitution of alanine at position 2 withMet, Asn, Gln, Leu, Ile, Phe, or Trp, the numbering of amino acidresidues being in accordance with that of SEQ ID NO: 1.

In some embodiments of the method, the polymerase is a Bst DNApolymerase comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a variantthereof having one or more conservative amino acid substitutions. Insome embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 2. In some embodiments, the disclosure relatesgenerally to an isolated variant of a protein comprising the amino acidsequence shown in SEQ ID NO: 2, wherein the variant comprises an aminoacid sequence that is at least 95% identical to SEQ ID NO: 2.

In some embodiments of the method, the polymerase is a Bst DNApolymerase comprising the amino acid sequence of SEQ ID NO: 3. In someembodiments, the disclosure relates generally to an isolated variant ofa protein comprising the amino acid sequence shown in SEQ ID NO: 3,wherein the variant comprises an amino acid sequence that is at least95% identical to SEQ ID NO: 3.

In some embodiments of the method, the polymerase is a Bst DNApolymerase comprising the amino acid sequence of SEQ ID NO: 4. In otherembodiments, the disclosure relates generally to an isolated variant ofa protein comprising the amino acid sequence shown in SEQ ID NO: 3,wherein the variant comprises an amino acid sequence that is at least95% identical to SEQ ID NO: 4.

In other embodiments of the method, the DNA polymerase is a Therminator™DNA polymerase comprising one or more conservative amino acidsubstitutions that substantially reduce its buffering capacity relativeto the corresponding wild-type protein within the range of about pH 7 toabout pH 9, wherein the one or more conservative amino acidsubstitutions are selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In other embodiments of the method, the DNA polymerase is a KOD DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 6.

In other embodiments of the method, the DNA polymerase is a B103 DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 7.

Candidate modified proteins may be synthesized in a variety of ways,including by way of molecular cloning and expression, or by chemicalsynthesis. Chemical synthesis of such proteins may be carried out bynative chemical ligation, as described by Dawson et al (Science 266:776-779 (1994)); Hakeng et al (Proc. Natl. Acad. Sci. USA 94: 7845-7850(1997)); Hakeng et al (Proc. Natl. Acad. Sci. USA 96: 10068-10073(1999)); and Kent et al., U.S. Pat. No. 6,184,344, which areincorporated herein by reference.

Modified proteins may also be produced by recombinant techniques. Insome embodiments, the disclosure relates generally to recombinant DNA orRNA molecules encoding a modified protein, including but not limited tophages, plasmids, phagemids, cosmids, YACs, BACs, as well as variousviral and non-viral vectors well known in the art, and cells transformedor transfected with such recombinant DNA or RNA molecules. Methods forgenerating such molecules are well known (see, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

In some embodiments, the disclosure relates generally to a host-vectorsystem comprising a recombinant DNA molecule containing a sequenceencoding a modified protein within a suitable prokaryotic or eukaryotichost cell. Examples of suitable eukaryotic host cells include a yeastcell, a plant cell, or an animal cell, such as a mammalian cell or aninsect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Polynucleotides comprising the coding sequence of a nonbuffering protein can be used to generate modified proteins using anynumber of host-vector systems routinely used and widely known in theart.

The disclosure relates generally to an isolated nucleic acid that is atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to a nucleic acid encoding any of the modifiedproteins disclosed herein.

Polynucleotide sequences encoding the modified proteins can be obtainedusing standard recombinant techniques. Alternatively, polynucleotidescan be synthesized using nucleotide synthesizer or PCR techniques. Onceobtained, sequences encoding the modified proteins are inserted into arecombinant vector capable of replicating and expressing heterologouspolynucleotides in prokaryotic hosts. Many vectors that are availableand known in the art can be used. Selection of an appropriate vectorwill depend mainly on the size of the nucleic acids to be inserted intothe vector and the particular host cell to be transformed with thevector. Each vector contains various components, depending on itsfunction (amplification or expression of heterologous polynucleotide, orboth) and its compatibility with the particular host cell in which itresides. The vector components generally include, but are not limitedto: an origin of replication, a selection marker gene, a promoter, aribosome binding site (RBS), a signal sequence, the heterologous nucleicacid insert and a transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector may comprise two or more promoter-cistron pairs,encoding each of the polypeptide components. A promoter is anuntranslated regulatory sequence located upstream (5′) to a cistron thatmodulates its expression. Prokaryotic promoters typically fall into twoclasses, inducible and constitutive. Inducible promoter is a promoterthat initiates increased levels of transcription of the cistron underits control in response to changes in the culture condition, e.g. thepresence or absence of a nutrient or a change in temperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector. Both the native promotersequence and many heterologous promoters may be used to directamplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby allowing a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In some embodiments, each cistron within the recombinant vectorcomprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a native part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected should be one that is recognizedand processed (i.e. cleaved by a signal peptidase) by the host cell. Forprokaryotic host cells that do not recognize and process the signalsequences native to the heterologous polypeptides, the signal sequenceis substituted by a prokaryotic signal sequence selected, for example,from the group consisting of the alkaline phosphatase, penicillinase,Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB,OmpA and MBP. In some embodiments, the signal sequences used in bothcistrons of the expression system are STII signal sequences or variantsthereof.

In some embodiments, the production of the modified proteins can occurin the cytoplasm of the host cell, and therefore does not require thepresence of secretion signal sequences within each cistron. Certain hoststrains (e.g., the E. coli trxB-strains) provide cytoplasm conditionsthat are favorable for disulfide bond formation, thereby permittingproper folding and assembly of expressed protein subunits. Proba andPluckthun Gene, 159:203 (1995).

Prokaryotic host cells suitable for expressing modified proteins includeArchaebacteria and Eubacteria, such as Gram-negative or Gram-positiveorganisms. Examples of useful bacteria include Escherichia (e.g., E.coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species(e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans,Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. Insome embodiments, gram-negative cells are used. In some embodiments, E.coli cells are used as hosts. Examples of E. coli strains include strainW3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington,D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCCDeposit No. 27,325) and derivatives thereof, including strain 33D3having genotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE)degP41 kanR (U.S. Pat. No. 5,639,635). Other strains and derivativesthereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coliλ 1776(ATCC 31,537) and E. coli RV308(ATCC 31,608) are also suitable. Theseexamples are illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce polypeptides according to thedisclosure can be grown in any suitable media, including media known inthe art and suitable for culture of the selected host cells. Examples ofsuitable media include luria broth (LB) plus necessary nutrientsupplements. In some embodiments, the media also contains a selectionagent, chosen based on the construction of the expression vector, toselectively permit growth of prokaryotic cells containing the expressionvector. For example, ampicillin is added to media for growth of cellsexpressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. Incertain embodiments, for E. coli growth, growth temperatures range fromabout 20° C. to about 39° C.; from about 25° C. to about 37° C.; orabout 30° C. The pH of the medium may be any pH ranging from about 5 toabout 9, depending mainly on the host organism. In certain embodiments,for E. coli, the pH is from about 6.8 to about 7.4, or about 7.0.

If an inducible promoter is used in the expression vector, proteinexpression is induced under conditions suitable for the activation ofthe promoter. In some embodiments, PhoA promoters are used forcontrolling transcription of the polypeptides. Accordingly, thetransformed host cells are cultured in a phosphate-limiting medium forinduction. In certain embodiments, the phosphate-limiting medium is theC.R.A.P. medium (see, e.g., Simmons et al., J. Immunol. Methods (2002),263:133-147). A variety of other inducers may be used, according to thevector construct employed, as is known in the art.

In some embodiments, the expressed polypeptides are secreted into andrecovered from the periplasm of the host cells. Protein recoverytypically involves disrupting the microorganism, generally by such meansas osmotic shock, sonication or lysis. Once cells are disrupted, celldebris or whole cells may be removed by centrifugation or filtration.The proteins may be further purified, for example, by affinity resinchromatography. Alternatively, proteins can be transported into theculture media and isolated therein. Cells may be removed from theculture and the culture supernatant being filtered and concentrated forfurther purification of the proteins produced. The expressedpolypeptides can be further isolated and identified using commonly knownmethods such as polyacrylamide gel electrophoresis (PAGE) and Westernblot assay.

In some embodiments, production of modified proteins is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, and in certain embodiments, about 1,000 to 100,000 liters ofcapacity. These fermentors use agitator impellers to distribute oxygenand nutrients, especially glucose (the preferred carbon/energy source).Small scale fermentation refers generally to fermentation in a fermentorthat is no more than approximately 100 liters in volumetric capacity,and can range from about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the proteins, variousfermentation conditions can be suitably modified. For example, toimprove the proper assembly and folding of the secreted modifiedproteins, additional vectors overexpressing chaperone proteins, such asDsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used. For example, host cellstrains may be modified to effect genetic mutation(s) in the genesencoding known bacterial proteases such as Protease III, OmpT, DegP,Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinationsthereof. Some E. coli protease-deficient strains are available anddescribed in, for example, Joly et al. (1998), supra; Georgiou et al.,U.S. Pat. No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Haraet al., Microbial Drug Resistance, 2:63-72 (1996).

In some embodiments, the modified protein includes one or more chemicalmodifications that reduce the buffering capacity of the protein. In oneexample, the modified protein includes an N-terminal amino acid residuethat is chemically modified, for example an N-terminal methionineresidue, by the addition of a formyl group. Such formylation can beuseful in reducing the buffering capacity of the N-terminal amino acidresidue that includes a primary amino group. Formyl-modified proteinscan be expressed and/or purified from host cells having a reducedactivity of methionine aminopeptidase (MAP) relative to a correspondingwild-type host cell. The use of such MAP-deficient strains can improveexpression of formyl-modified proteins. See, e.g., Sherman, F., J. W.Stewart, and S. Tsunasawa. 1985. Methionine or not methionine at thebeginning of a protein. BioEssays 3:27-31.

In some embodiments, the modified protein produced herein is furtherpurified to obtain preparations that are substantially homogeneous forfurther assays and uses. Standard protein purification methods known inthe art can be employed. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns, ethanol precipitation, reverse phase HPLC,chromatography on silica or on a cation-exchange resin such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using, for example, Sephadex G-75.

In some embodiments, the disclosure relates generally to polypeptidescomprising a modified protein fused to another polypeptide to form achimeric polypeptide. In some embodiments, the chimeric polypeptidecomprises a tag polypeptide which provides an epitope which isselectively bound by an anti-tag antibody. The epitope tag allows themodified protein to be purified by affinity purification using theanti-tag antibody. Epitope tags and their respective antibodies are wellknown in the art. Examples include the flu HA tag polypeptide and itsantibody 12CA5 (Field et al. Mol. Cell Biol. 8: 2159-2165 (1988)); thec-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto(Evan et al., Mol. Cell Biol. 5: 3610-3616 (1985); and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al.,Protein Engineering 3: 547-553 (1990)). Other examples of epitope tagsinclude the Flag-peptide (Hopp et al., BioTechnology 6: 1204-1210(1998); the KT3 epitope peptide (Martin et al., Science 255: 192-194(1992)); an a-tubulin epitope peptide (Skinner et al, J. Biol. Chem.266: 15163-15166 (1991)) and the T7 gp10 peptide tag (Lutz-Freyermuth etal., Proc. Natl. Acad. Sci. USA 87: 6393-6397 (1990)).

Epitope-tagged modified proteins can be conveniently purified byaffinity chromatography using the anti-tag antibody. The matrix to whichthe anti-tag antibody is attached is most often agarose, but othermatrices are available (e.g., controlled pore glass orpoly(styrenedivinyl)benzene). The epitope-tagged modified proteins canbe eluted from the affinity column by, for example, varying the bufferpH or ionic strength, or by addition of chaotropic agents.

In some embodiments, a modified protein is fused to a histidine tag(His-tag). The His-tagged modified protein can be purified by, forexample, using a nickel column using standard techniques.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits that may be used for thedetection and/or monitoring of biological and chemical reactions. Thesereactions may include enzymatic reactions in which substrates and/orreagents are consumed and/or reaction intermediates, byproducts and/orproducts are generated. In some embodiments, the disclosure relatesgenerally to compositions and methods that may be used for the detectionand/or monitoring of biological and chemical reactions wherein theconcentration of at least one type of ion changes during the course ofthe reaction. An example of such a reaction is a nucleic acid synthesismethod such as one that provides information regarding nucleic acidsequence.

In various embodiments, a biological or chemical reaction is detectedand/or monitored by a sensor including a field-effect transistor (FET).In various embodiments the FET is a chemFET or an ISFET. A “chemFET” orchemical field-effect transistor, is a type of field effect transistorthat acts as a chemical sensor. It is the structural analog of a MOSFETtransistor, where the charge on the gate electrode is applied by achemical process. An “ISFET” or ion-sensitive field-effect transistor,is used for measuring ion concentrations in solution; when the ionconcentration (such as H+) changes, the current through the transistorwill change accordingly. A detailed theory of operation of an ISFET isgiven in “Thirty years of ISFETOLOGY: what happened in the past 30 yearsand what may happen in the next 30 years,” P. Bergveld, Sens. Actuators,88 (2003), pp. 1-20.

In some embodiments, the FET may be a FET array. As used herein, an“array” is a planar arrangement of elements such as sensors or wells.The array may be one or two dimensional. A one dimensional array is anarray having one column (or row) of elements in the first dimension anda plurality of columns (or rows) in the second dimension. The number ofcolumns (or rows) in the first and second dimensions may or may not bethe same. The FET or array may comprise 102, 103, 104, 105, 106, 107 ormore FETs.

In some embodiments, one or more microfluidic structures is/arefabricated above the FET sensor array to provide for containment and/orconfinement of a biological or chemical reaction. For example, in oneimplementation, the microfluidic structure(s) may be configured as oneor more wells (or microwells, or reaction chambers, or reaction wells,as the terms are used interchangeably herein) disposed above one or moresensors of the array, such that the one or more sensors over which agiven well is disposed detect and measure analyte presence, level,and/or concentration in the given well. In some embodiments, there is a1:1 correspondence of FET sensors and reaction wells.

Microwells or reaction chambers are typically hollows or wells havingwell-defined shapes and volumes which are manufactured into a substrateand may be fabricated using conventional microfabrication techniques,e.g. as disclosed in the following references: Doering and Nishi,Editors, Handbook of Semiconductor Manufacturing Technology, SecondEdition (CRC Press, 2007); Saliterman, Fundamentals of BioMEMS andMedical Microdevices (SPIE Publications, 2006); Elwenspoek et al,Silicon Micromachining (Cambridge University Press, 2004); and the like.Preferable configurations (e.g. spacing, shape and volumes) ofmicrowells or reaction chambers are disclosed in Rothberg et al, U.S.patent publication 2009/0127589; Rothberg et al, U.K. patent applicationGB24611127, which are incorporated by reference.

In some embodiments, the biological or chemical reaction is performed ina solution or a reaction chamber that is in contact with or capacitivelycoupled to a FET such as a chemFET or an ISFET. The FET (or chemFET orISFET) and/or reaction chamber may be an array of FETs or reactionchambers, respectively.

In some embodiments, a biological or chemical reaction is carried out ina two-dimensional array of reaction chambers, wherein each reactionchamber is coupled to a FET, and each reaction chamber is no greaterthan 10 μm³ (i.e., 1 pL) in volume. In some embodiments each reactionchamber is no greater than 0.34 pL, 0.096 pL or even 0.012 pL in volume.A reaction chamber can optionally be 22, 32, 42, 52, 62, 72, 82, 92, or102 square microns in cross-sectional area at the top. Preferably, thearray has at least 102, 103, 104, 105, 106, 107, 108, 109, or morereaction chambers. In some embodiments, the reaction chambers arecapacitively coupled to the FETs.

FET arrays as used in various embodiments according to the disclosuremay be fabricated according to conventional CMOS fabricationstechniques, as well as modified CMOS fabrication techniques and othersemiconductor fabrication techniques beyond those conventionallyemployed in CMOS fabrication. Additionally, various lithographytechniques may be employed as part of an array fabrication process.

Exemplary FET arrays suitable for use in the disclosed methods, as wellas microwells and attendant fluidics, and methods for manufacturingthem, are disclosed, for example, in U.S. Patent Publication No.20100301398; U.S. Patent Publication No. 20100300895; U.S. PatentPublication No. 20100300559; U.S. Patent Publication No. 20100197507,U.S. Patent Publication No. 20100137143; U.S. Patent Publication No.20090127589; and U.S. Patent Publication No. 20090026082.

In some embodiments, the disclosure relates generally to a method ofdetecting a change in ion concentration during a chemical reaction,comprising: performing a chemical reaction in the presence of a modifiedpolypeptide having one or more amino acid substitutions, wherein theconcentration of at least one type of ion changes during the course ofthe chemical reaction; and detecting a signal indicating the change inion concentration, wherein the signal is increased relative to a signalthat is detected from a chemical reaction performed in the presence ofthe unmodified polypeptide but under otherwise identical reactionconditions.

In some embodiments, the modified polypeptide includes one or more aminoacid substitutions that reduce the buffering capacity of the modifiedpolypeptide relative to the unmodified polypeptide.

In some embodiments, at least one of the one or more amino acidsubstitutions includes the substitution of an amino acid residue havinga pKa of between about 4 and 10 with another amino acid residue having apKa less than about 4 or greater than about 10.

In some embodiments, the modified polypeptide is a DNA or RNA bindingprotein. In various embodiments, the polypeptide is a DNA polymerase, ahelicase, a ligase, a nuclease, a single stranded DNA binding protein ora polymerase accessory protein.

In some embodiments, the modified polypeptide is a modified polymerase,and the chemical reaction includes a nucleotide incorporation.

In some embodiments of the method, the modified polypeptide is apolymerase comprising one or more amino acid substitutions that reducethe buffering capacity of said protein relative to the correspondingwild-type protein within the range of about pH 4 to about pH 10. In someembodiments, the one or more amino acid substitutions reduce thebuffering capacity of the polymerase relative to the correspondingwild-type polymerase within the range of about pH 7 to about pH 9.

In some embodiments, the one or more amino acid substitutionssubstantially reduce the buffering capacity of the polymerase within therange of about pH 7 to pH 9. In some embodiments, at least one of theone or more amino acid substitution is a conservative amino acidsubstitution. In further embodiments, the at least one conservativeamino acid substitution is selected from the group consisting ofhistidine to arginine, glutamic acid to glutamine, aspartic acid toasparagine, lysine to arginine, and tyrosine to phenylalanine.

In various embodiments, the DNA polymerase is selected from the groupconsisting of an A family DNA polymerase; a B family DNA polymerase; amixed-type polymerase; an unclassified DNA polymerase and RT familypolymerase; and variants and derivatives thereof.

In some embodiments, the DNA polymerase is an A family DNA polymeraseselected from the group consisting of a Pol I-type DNA polymerase suchas E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, Bst DNA polymerase, Taq DNA polymerase, Platinum Taq DNApolymerase series, T7 DNA polymerase, and Tth DNA polymerase. In someembodiments, the DNA polymerase is Bst DNA polymerase. In otherembodiments, the DNA polymerase is E. coli DNA polymerase. In someembodiments, the DNA polymerase is the Klenow fragment of E. coli DNApolymerase. In some embodiments, the polymerase is Taq DNA polymerase.In some embodiments, the polymerase is T7 DNA polymerase.

In other embodiments, the DNA polymerase is a B family DNA polymeraseselected from the group consisting of Bst polymerase, Tli polymerase,Pfu polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase,KOD polymerase, Sac polymerase, Sso polymerase, Poc polymerase, Pabpolymerase, Mth polymerase, Pho polymerase, ES4 polymerase, VENTpolymerase, DEEPVENT polymerase, Therminator™ polymerase, phage Phi29polymerase, and phage B103 polymerase. In some embodiments, thepolymerase is KOD polymerase. In some embodiments, the polymerase isTherminator™ polymerase. In some embodiments, the polymerase is phagePhi29 DNA polymerase. In some embodiments the polymerase is phage B103polymerase, including, for example, the variants disclosed in U.S.Patent Publication No. 20110014612 which is incorporated by referenceherein.

In other embodiments, the DNA polymerase is a mixed-type polymeraseselected from the group consisting of EX-Taq polymerase, LA-Taqpolymerase, Expand polymerase series, and Hi-Fi polymerase. In yet otherembodiments, the DNA polymerase is an unclassified DNA polymeraseselected from the group consisting of Tbr polymerase, Tfl polymerase,Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tihpolymerase, and Tfi polymerase.

In other embodiments, the DNA polymerase is an RT polymerase selectedfrom the group consisting of HIV reverse transcriptase, M-MLV reversetranscriptase and AMV reverse transcriptase. In some embodiments, thepolymerase is HIV reverse transcriptase or a fragment thereof having DNApolymerase activity.

In some embodiments of the method, the DNA polymerase is a Bst DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 2 or Table 3. Insome embodiments, the one or more conservative amino acid substitutionsare selected from the group consisting of H46R, H273R, H281R, E446Q,H473R, H528R, H572R and Y477F, the numbering of amino acid residuesbeing in accordance with that of SEQ ID NO: 1.

In some embodiments, the Bst DNA polymerase comprises one or moreconservative amino acid substitutions, wherein the one or more aminoacid substitutions includes a substitution of alanine at position 2 withMet, Asn, Gln, Leu, Ile, Phe, or Trp, the numbering of amino acidresidues being in accordance with that of SEQ ID NO: 1.

In some embodiments of the method, the polymerase is a Bst DNApolymerase comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a variantthereof having one or more conservative amino acid substitutions. Insome embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 2. In some embodiments, the disclosure relatesgenerally to an isolated variant of a protein comprising the amino acidsequence shown in SEQ ID NO: 2, wherein the variant comprises an aminoacid sequence that is at least 95% identical to SEQ ID NO: 2.

In some embodiments, the polymerase is a Bst DNA polymerase comprisingthe amino acid sequence of SEQ ID NO: 3. In some embodiments, thedisclosure relates generally to an isolated variant of a proteincomprising the amino acid sequence shown in SEQ ID NO: 3, wherein thevariant comprises an amino acid sequence that is at least 95% identicalto SEQ ID NO: 3.

In some embodiments, the polymerase is a Bst DNA polymerase comprisingthe amino acid sequence of SEQ ID NO: 4. In other embodiments, thedisclosure relates generally to an isolated variant of a proteincomprising the amino acid sequence shown in SEQ ID NO: 3, wherein thevariant comprises an amino acid sequence that is at least 95% identicalto SEQ ID NO: 4.

In other embodiments of the method, the DNA polymerase is a Therminator™DNA polymerase comprising one or more conservative amino acidsubstitutions that substantially reduce its buffering capacity relativeto the corresponding wild-type protein within the range of about pH 7 toabout pH 9, wherein the one or more conservative amino acidsubstitutions are selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In other embodiments of the method, the DNA polymerase is a KOD DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 6.

In other embodiments of the method, the DNA polymerase is a B103 DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 7.

In some embodiments of the method, the polymerase comprises one or moreamino acid substitutions that reduce the buffering capacity of saidprotein relative to the corresponding wild-type protein within the rangeof about pH 4 to about pH 10, wherein at least one of the one or moreamino acid substitutions includes a substitution of an amino acidresidue having a pKa within the range of about 4.0 to about 10.0 withanother amino acid residue. In some embodiments, the pKa of the aminoacid residue is a solution pKa of the amino acid residue. In otherembodiments, the pKa of the amino acid residue is a pKa of the aminoacid residue in the context of the corresponding wild-type protein.

In some embodiments, at least one of the one or more amino substitutionsincludes a substitution of an amino acid residue having a pKa of betweenabout 4.0 and about 10.0 with an amino acid residue having a pKa that isgreater than about 10.0 or less than about 4.0. In further embodimentsthe amino acid residue having a pKa that is greater than about 10.0 orless than about 4.0 is selected from the group consisting of: Arg, Asp,Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp, Val andN-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 6.0 to about 8.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidmodifications includes a substitution of an amino acid residue having apKa of between about 6.0 and about 8.0 with an amino acid residue havinga pKa that is greater than about 8.0 or less than about 6.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue selectedfrom the group consisting of His, Glu, Asp, Tyr, and Lys with anotheramino acid residue.

In some embodiments, at least one of the one or more amino acidmodifications includes a substitution of an amino acid residue with analanine residue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue that isat least 30% solvent exposed in the corresponding wild-type protein withanother amino acid residue.

In some embodiments of the method, the polymerase comprises one or morechemical amino acid modifications that reduce the buffering capacity ofsaid protein relative to the corresponding wild-type protein within therange of about pH 4 to about pH 10. In some embodiments, the one or morechemical amino acid modifications includes a chemical modification ofthe N-terminal amino acid. In some embodiments, the one or more chemicalamino acid modifications includes a chemical modification of an aminoacid residue including a primary amine group with an amine-reactiveagent. In some embodiments, the amine-reactive reagent includes anacylating agent or an activated ester.

In some embodiments of the method of detecting a change in ionconcentration during a chemical reaction, the detecting step comprisesusing a chemFET. In some embodiments, the chemFET is an ISFET. In someembodiments, the ISFET is in an ISFET array.

In some embodiments, the chemical reaction is performed in a reactionchamber comprising or capacitively coupled to a chemFET.

In some embodiments, the chemical reaction is in the presence of anagent that alters the value of a pKa of at least an amino acid in themodified polypeptide from within the range of about 6 to about 8 to lessthan about 6 or greater than about 8. In some embodiments, the agent isa phospholipid, a sulfonic acid surfactant, a polyanionic electrolyte ora salt thereof, a polycationic electrolyte or a salt thereof,tetramethyl ammonium or a salt thereof, or a combination thereof.

In some embodiments, at least one type of ion is hydrogen ion.

In some embodiments, the reaction is an enzymatic reaction or a bindingreaction.

In some embodiments, the chemical reaction is an enzymatic reaction. Insome embodiments, the enzymatic reaction is a nucleotide incorporationreaction.

In some embodiments, the disclosed methods further include repeatingsteps a)-b).

In some embodiments, the disclosure relates generally to methods forusing the modified proteins of the disclosure to obtain sequenceinformation regarding a nucleic acid of interest. Such methods involvethe synthesis of a new nucleic acid (e.g., using a primer that ishybridized to a template nucleic acid or a self-priming template, aswill be appreciated by those of ordinary skill), based on the sequenceof a template nucleic acid. That is, the sequence of the newlysynthesized nucleic acid is complementary to the sequence of thetemplate nucleic acid and therefore knowledge of sequence of the newlysynthesized nucleic acid yields information about the sequence of thetemplate nucleic acid.

In one aspect, the disclosed methods, compositions, systems, apparatusesand kits may be used for carrying out label-free nucleic acidsequencing, and in particular, ion-based nucleic acid sequencing. Theconcept of label-free nucleic acid sequencing, including ion-basednucleic acid sequencing, has been described in the literature, includingthe following references that are incorporated by reference: Rothberg etal, U.S. patent publication 2009/0026082; Anderson et al, Sensors andActuators B Chem., 129: 79-86 (2008); and Pourmand et al, Proc. Natl.Acad. Sci., 103: 6466-6470 (2006). Briefly, in nucleic acid sequencingapplications, nucleotide incorporations are determined by measuringnatural byproducts of polymerase-catalyzed extension reactions,including hydrogen ions, polyphosphates, PPi, and Pi (e.g., in thepresence of pyrophosphatase).

In a typical embodiment of ion-based nucleic acid sequencing, nucleotideincorporations are detected by detecting the presence and/orconcentration of hydrogen ions generated by polymerase-catalyzedextension reactions. In one embodiment, templates each having a primerand polymerase operably bound are loaded into reaction chambers (such asthe microwells disclosed in Rothberg et al, cited above), after whichrepeated cycles of nucleotide addition and washing are carried out. Insome embodiments, such templates may be attached as clonal populationsto a solid support, such as a microparticle, bead, or the like, and saidclonal populations are loaded into reaction chambers. For example,templates may be prepared as disclosed in U.S. Pat. No. 7,323,305, whichis incorporated by reference. As used herein, “operably bound” meansthat a primer is annealed to a template so that the primer's 3′ end maybe extended by a polymerase and that a polymerase is bound to suchprimer-template duplex, or in close proximity thereof so that bindingand/or extension takes place whenever nucleotides are added.

In each addition step of the cycle, the polymerase extends the primer byincorporating added nucleotide only if the next base in the template isthe complement of the added nucleotide. If there is one complementarybase, there is one incorporation, if two, there are two incorporations,if three, there are three incorporations, and so on. With each suchincorporation there is a hydrogen ion released, and collectively apopulation of templates releasing hydrogen ions changes the local pH ofthe reaction chamber. The production of hydrogen ions is monotonicallyrelated to the number of contiguous complementary bases in the template(as well as the total number of template molecules with primer andpolymerase that participate in an extension reaction). Thus, when thereare a number of contiguous identical complementary bases in the template(i.e. a homopolymer region), the number of hydrogen ions generated, andtherefore the magnitude of the local pH change, is proportional to thenumber of contiguous identical complementary bases. If the next base inthe template is not complementary to the added nucleotide, then noincorporation occurs and no hydrogen ion is released. In someembodiments, after each step of adding a nucleotide, an additional stepmay be performed, in which an unbuffered wash solution at apredetermined pH is used to remove the nucleotide of the previous stepin order to prevent misincorporations in later cycles. In someembodiments, the after each step of adding a nucleotide, an additionalstep may be performed wherein the reaction chambers are treated with anucleotide-destroying agent, such as apyrase, to eliminate any residualnucleotides remaining in the chamber, which may result in spuriousextensions in subsequent cycles.

In one exemplary embodiment, different kinds of nucleotides are addedsequentially to the reaction chambers, so that each reaction is exposedto the different nucleotides one at a time. For example, nucleotides canbe added in the following sequence: dATP, dCTP, dGTP, dTTP, dATP, dCTP,dGTP, dTTP, and so on; with each exposure followed by a wash step. Thecycles may be repeated for 50 times, 100 times, 200 times, 300 times,400 times, 500 times, 750 times, or more, depending on the length ofsequence information desired.

In some embodiments, the polymerase can include without limitation anyenzyme that can catalyze the polymerization of nucleotides (includinganalogs thereof) into a nucleic acid strand. Typically but notnecessarily such nucleotide polymerization can occur in atemplate-dependent fashion. Such polymerases can include withoutlimitation naturally occurring polymerases and any subunits andtruncations thereof, mutant polymerases, variant polymerases,recombinant, fusion or otherwise engineered polymerases, chemicallymodified polymerases, synthetic molecules or assemblies, and anyanalogs, derivatives or fragments thereof that retain the ability tocatalyze such polymerization. Optionally, the polymerase can be a mutantpolymerase comprising one or more mutations involving the replacement ofone or more amino acids with other amino acids, the insertion ordeletion of one or more amino acids from the polymerase, or the linkageof parts of two or more polymerases. Typically, the polymerase comprisesone or more active sites at which nucleotide binding and/or catalysis ofnucleotide polymerization can occur. Some exemplary polymerases includewithout limitation DNA polymerases (such as for example E. coli DNApolymerase, Bst DNA polymerase, Taq polymerase, T7 polymerase, Phi-29DNA polymerase, B103 polymerase and reverse transcriptases) and RNApolymerases. The term “polymerase” and its variants may also refer tofusion proteins comprising at least two portions linked to each other,where the first portion comprises a peptide that can catalyze thepolymerization of nucleotides into a nucleic acid strand and is linkedto a second portion that comprises a second polypeptide, such as, forexample, a reporter enzyme or a processivity-enhancing domain. Oneexemplary embodiment of such a polymerase is Phusion® DNA polymerase(New England Biolabs), which comprises a Pyrococcus-like polymerasefused to a processivity-enhancing domain as described, for example, inU.S. Pat. No. 6,627,424.

In some embodiments, the modified polymerase comprises one or moremodifications resulting in altered exonuclease activity (for example 3′to 5′ exonuclease activity) as compared to a reference polymerase (forexample, an unmodified counterpart). In some embodiments, themodification comprises an amino acid substitution. In some embodiments,the modified polymerase lacks 3′ to 5′ exonuclease activity, or lacks 5′to 3′ exonuclease activity, or both. Mutations at conversed amino acidresidues that reduce or eliminate 3′ to 5′ exonuclease activity havebeen described for various polymerases. For example, amino acidmutations reducing exonuclease activity in Phi-29-type polymerases atvarious residues have been described, for example, in de Vega et al.,“Primer-terminus stabilization at the 3′-5′ exonuclease active site ofΦ29 DNA polymerase. Involvement of two amino acid residues highlyconserved in proofreading DNA polymerases” EMBO J., 15(5):1182-1192(1996); Soengas et al., “Site-directed mutagenesis at the Exo III motifof D29 DNA polymerase; overlapping structural domains for the 3′-5′exonuclease and strand-displacement activities” EMBO J.,11(11):4227-4237 (1992); Blanco et al., U.S. Pat. Nos. 5,001,050,5,198,543 and 5,576,204.

One exemplary polymerase suitable for use in some but not allembodiments is the exo-minus (exo-) version of the Klenow fragment of E.coli DNA polymerase I which lacks 3′ to 5′ exonuclease activity. Otherpolymerases include T4 exo-, Therminator™, and Bst polymerases, as wellas variants of the B106 DNA polymerase containing modifications thatreduce its 3′ to 5′ exonuclease activity as disclosed in U.S. PatentPublication No. 20110014612, which is incorporated by reference herein.In still other embodiments that require excision of nucleotides (e.g.,in the process of a nick translation reaction), polymerases withexonuclease activity are preferred. Since DNA synthesis fidelity dependsmainly on the presence or absence of 3′-5′ exonuclease activity, enzymeswith 3′-5′ exonuclease activity can be used in some embodiments. Acombination of enzymes can also be used. The polymerase may be one thatis modified to comprise accessory factors including without limitationsingle or double stranded DNA binding proteins.

Some embodiments may require that the polymerase have sufficientprocessivity. As used herein, processivity is the ability of apolymerase to remain bound to a single primer/template hybrid. It may bemeasured by the number of nucleotides that a polymerase incorporatesinto a nucleic acid (such as a sequencing primer) prior to dissociationof the polymerase from the primer/template hybrid. In some embodiments,the polymerase has a processivity of at least 100 nucleotides, althoughin other embodiments it has a processivity of at least 200 nucleotides,at least 300 nucleotides, at least 400 nucleotides, or at least 500nucleotides. It will be understood by those of ordinary skill in the artthat the higher the processivity of the polymerase, the more nucleotidesthat can be incorporated prior to dissociation, and therefore the longerthe sequence that can be obtained. In other words, polymerases havinglow processivity will provide shorter read-lengths than will polymeraseshaving higher processivity. As an example, a polymerase that dissociatesfrom the hybrid after five incorporations will only provide a sequenceof 5 nucleotides in length, while a polymerase that dissociates onaverage from the hybrid after 500 incorporations will provide sequenceof about 500 nucleotides.

The rate at which a polymerase incorporates nucleotides will varydepending on the particular application, although generally faster ratesof incorporation are desirable. The rate of “sequencing” will depend onthe number of arrays on chip, the size of the wells, the temperature andconditions at which the reactions are run, etc.

In some embodiments, the nucleotides are delivered at substantially thesame time to each template. In some embodiments, polymerase(s) arealready present, although in some embodiments they may be introducedalong with the nucleotides. The polymerases may be immobilized or may befree flowing. In embodiments where the polymerases are immobilized, thepolymerases may be attached to a solid support such as a bead surface, abead interior or some combination of bead surface and interior.Typically, the bead is present in a reaction chamber, although themethods may also be carried out in the absence of reaction chambers. Thesolid support may also be the sensor surface or a wall of a reactionchamber that is capacitively coupled to the sensor.

The reaction may occur in a reaction chamber in some embodiments, whilein others it may occur in the absence of reaction chambers. In theselatter embodiments, the sensor surface may be continuous without anyphysical divider between sensors.

In some embodiments, the nucleotide can include any compound that canbind selectively to, or can be polymerized by, a polymerase. Typically,but not necessarily, selective binding of the nucleotide to thepolymerase is followed by polymerization of the nucleotide into anucleic acid strand by the polymerase; occasionally however thenucleotide may dissociate from the polymerase without becomingincorporated into the nucleic acid strand, an event referred to hereinas a “non-productive” event. The nucleotide can include not only anynaturally occurring nucleotide but also any analog, regardless of itsstructure, that can bind selectively to, or can be polymerized by, apolymerase. While naturally occurring nucleotides typically comprisebase, sugar and phosphate moieties, the nucleotides of the presentdisclosure can include compounds lacking any one, some or all of suchmoieties. In some embodiments, the nucleotide can optionally include achain of phosphorus atoms comprising three, four, five, six, seven,eight, nine, ten or more phosphorus atoms. In some embodiments, thephosphorus chain can be attached to any carbon of a sugar ring, such asthe 5′ carbon. The phosphorus chain can be linked to the sugar with anintervening O or S. In one embodiment, one or more phosphorus atoms inthe chain can be part of a phosphate group having P and O. In anotherembodiment, the phosphorus atoms in the chain can be linked togetherwith intervening O, NH, S, methylene, substituted methylene, ethylene,substituted ethylene, CNH2, C(O), C(CH2), CH2CH2, or C(OH)CH2R (where Rcan be a 4-pyridine or 1-imidazole). In one embodiment, the phosphorusatoms in the chain can have side groups having O, BH3, or S. In thephosphorus chain, a phosphorus atom with a side group other than O canbe a substituted phosphate group. In the phosphorus chain, phosphorusatoms with an intervening atom other than O can be a substitutedphosphate group. Some examples of nucleotide analogs are described inXu, U.S. Pat. No. 7,405,281. In some embodiments, the nucleotidecomprises a label and referred to herein as a “labeled nucleotide”. Insome embodiments, the label can be in the form of a fluorescent dyeattached to the terminal phosphate group, i.e., the phosphate group mostdistal from the sugar. Some examples of nucleotides that can be used inthe disclosed methods and compositions include, but are not limited to,ribonucleotides, deoxyribonucleotides, modified ribonucleotides,modified deoxyribonucleotides, ribonucleotide polyphosphates,deoxyribonucleotide polyphosphates, modified ribonucleotidepolyphosphates, modified deoxyribonucleotide polyphosphates, peptidenucleotides, modified peptide nucleotides, metallonucleosides,phosphonate nucleosides, and modified phosphate-sugar backbonenucleotides, analogs, derivatives, or variants of the foregoingcompounds, and the like. In some embodiments, the nucleotide cancomprise non-oxygen moieties such as, for example, thio- orborano-moieties, in place of the oxygen moiety bridging the alphaphosphate and the sugar of the nucleotide, or the alpha and betaphosphates of the nucleotide, or the beta and gamma phosphates of thenucleotide, or between any other two phosphates of the nucleotide, orany combination thereof.

Some embodiments of the disclosure relate generally to detectinghydrogen ions released as a function of nucleotide incorporation; someembodiments relate generally to detecting hydrogen ions released as afunction of nucleotide excision. It is important in these and variousother aspects to detect as many released hydrogen ions as possible inorder to achieve as high a signal (and/or a signal to noise ratio) aspossible. Strategies for increasing the number of released protons thatare ultimately detected by the FET surface include without limitationlimiting interaction of released protons with reactive groups in thewell, choosing a material from which to manufacture the well in thefirst instance that is relatively inert to protons, preventing releasedprotons from exiting the well prior to detection at the chemFET, andincreasing the copy number of templates per well (in order to amplifythe signal from each nucleotide incorporation), among others.

Some embodiments of the disclosed methods employ an environment, forexample a reaction solution, which is minimally buffered, if at all.Buffering can be contributed by the components of the solution or by thesolid supports in contact with such solution. A solution having no orlow buffering capacity (or activity) is one in which changes in hydrogenion concentration on the order of at least about +/−0.005 pH units, atleast about +/−0.01, at least about +/−0.015, at least about +/−0.02, atleast about +/−0.03, at least about +/−0.04, at least about +/−0.05, atleast about +/−0.10, at least about +/−0.15, at least about +/−0.20, atleast about +/−0.25, at least about +/−0.30, at least about +/−0.35, atleast about +/−0.45, at least about +/−0.50, or more are detectable(e.g., using the chemFET sensors described herein). In some embodiments,the pH change per nucleotide incorporation is on the order of about0.005. In some embodiments, the pH change per nucleotide incorporationis a decrease in pH. Reaction solutions that have no or low bufferingcapacity may contain no or very low concentrations of buffer, or may useweak buffers.

The reaction solution may have a buffer concentration equal to or lessthan 1 mM, equal to or less than 0.9 mM, equal to or less than 0.8 mM,equal to or less than 0.7 mM, equal to or less than 0.6 mM, equal to orless than 0.5 mM, equal to or less than 0.4 mM, equal to or less than0.3 mM, equal to or less than 0.2 mM, equal to or less than 0.1 mM, orless including zero. The buffer concentration may be 50-100 μM. Anon-limiting example of a weak buffer suitable for the sequencingreactions described herein wherein pH change is the readout is 0.1 mMTris or Tricine.

In some aspects, in addition to or instead of using reduced bufferingsolutions, nucleotide incorporation (and optionally excision) is carriedout in the presence of additional agents that serve to shield potentialbuffering events that may occur in solution. These agents are referredto herein as buffering inhibitors since they inhibit the ability ofcomponents within a solution or a solid support in contact with thesolution to sequester and/or otherwise interfere with released hydrogenions prior to their detection by the FET surface. In the absence of suchinhibitors, released hydrogen ions may interact with or be sequesteredby reactive groups in the solution or on solid supports in contact withthe solution. These hydrogen ions are less likely to reach and bedetected by the FET surface, leading to a weaker signal than isotherwise possible. In the presence of such inhibitors however therewill be fewer reactive groups available for interaction with orsequestration of hydrogen ions. As a result, a greater proportion ofreleased hydrogen ions will reach and be detected by the FET surface,leading to stronger signals. Some suitable buffering inhibitorsdemonstrate little or no buffering capacity in the pH range of 5-9,meaning that pH changes on the order of 0.005, 0.01, 0.02, 0.03, 0.04,0.05, 0.1, 0.2, 0.3, 0.4, 0.5 or more pH units are detectable (e.g., byusing an ISFET) in the presence of such inhibitors.

There are various types of buffering inhibitors. One example of a classof buffering inhibitors is phospholipids. The phospholipids may benaturally occurring or non-naturally occurring phospholipids. Examplesof phospholipids that may be used as buffering inhibitors include butare not limited to phosphatidylcholine, phosphatidylethanolamine,phosphatidylglycerol, and phosphatidylserine.

Another example of a class of buffering inhibitors is sulfonic acidbased surfactants such as poly(ethylene glycol) 4-nonylphenyl3-sulfopropyl ether (PNSE). In addition to shielding reactive groupsthat would otherwise interfere with released protons, PNSE has also beenreported to enhance polymerase activity.

Another example of a class of buffering inhibitors is polyanionicelectrolytes such as poly(styrenesulfonic acid or its sodium salt.

Another example of a class of buffering inhibitors is polycationicelectrolytes such as poly(diallydimethylammonium) or its chloride salt.These compounds are known to bind to DNA.

Another example of a buffering inhibitor is tetramethyl ammonium or itschloride salt.

These various inhibitors may be present throughout a reaction by beingincluded in nucleotide solutions, wash solutions, and the like.Alternatively, they may be flowed through the reaction chamber at settimes relative to the flow through of nucleotides and/or other reactionreagents. In still other embodiments, they may be coated on the FETsurface (or reaction chamber surface). Such coating may be covalent ornon-covalent.

In one embodiment, the disclosure relates generally to a method forobtaining sequence information from a nucleic acid template, comprising:(a) providing a template nucleic acid hybridized to a sequencing primerand bound to a polymerase, wherein the polymerase comprises one or moreamino acid substitutions that substantially reduce its bufferingcapacity within the range of about pH 4 to about pH 10; (b) synthesizinga new nucleic acid strand by sequentially incorporating one or morenucleotides at the 3′ end of the sequencing primer, wherein a hydrogenion byproduct is generated when the nucleotide is complementary tocorresponding nucleotides in the template nucleic acid; and (c)detecting the incorporation of the one or more nucleotides into thesequencing primer by detecting the release of hydrogen ions.

In some embodiments, the one or more amino acid substitutions in thepolymerase reduce the buffering capacity of the polymerase relative tothe corresponding wild-type (unsubstituted) polymerase within the rangeof about pH 7 to about pH 9.

In some embodiments, at least one of the one or more amino acidsubstitutions in the polymerase is a conservative amino acidsubstitution. In further embodiments, the at least one conservativeamino acid substitution is selected from the group consisting ofhistidine to arginine, glutamic acid to glutamine, aspartic acid toasparagine, lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, the polymerase comprises one or more amino acidsubstitutions that reduce the buffering capacity of said polymeraserelative to the corresponding wild-type polymerase within the range ofabout pH 4 to about pH 10, wherein at least one of the one or more aminoacid substitutions includes a substitution of an amino acid residuehaving a pKa within the range of about 4.0 to about 10.0 with anotheramino acid residue. In some embodiments, the pKa of the amino acidresidue is a solution pKa of the amino acid residue. In otherembodiments, the pKa of the amino acid residue is a pKa of the aminoacid residue in the context of the corresponding wild-type protein.

In some embodiments, at least one of the one or more amino substitutionsincludes a substitution of an amino acid residue having a pKa of betweenabout 4.0 and about 10.0 with an amino acid residue having a pKa that isgreater than about 10.0 or less than about 4.0. In further embodimentsthe amino acid residue having a pKa that is greater than about 10.0 orless than about 4.0 is selected from the group consisting of: Arg, Asp,Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp, Val andN-terminal Formylmethionine (N-fMet).

In some embodiments, the polymerase comprises one or more amino acidsubstitutions that reduce the buffering capacity of said polymeraserelative to the corresponding wild-type polymerase within the range ofabout pH 7 to about pH 9. In some embodiments, at least one of the oneor more amino acid substitutions includes a substitution of an aminoacid residue having a pKa within the range of about 7 to about 9 withanother amino acid residue. In some embodiments, the pKa of the aminoacid residue is a solution pKa of the amino acid residue. In otherembodiments, the pKa of the amino acid residue is a pKa of the aminoacid residue in the context of the corresponding wild-type protein.

In some embodiments, at least one of the one or more amino substitutionsincludes a substitution of an amino acid residue having a pKa of betweenabout 7 and about 9 with an amino acid residue having a pKa that is lessthan about 7 or greater than about 9. In further embodiments the aminoacid residue having a pKa that is greater than about 10.0 or less thanabout 4.0 is selected from the group consisting of: Arg, Asp, Gln, ly,Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp, Val and N-terminalFormylmethionine (N-fMet).

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 6.0 to about 8.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidmodifications includes a substitution of an amino acid residue having apKa of between about 6.0 and about 8.0 with an amino acid residue havinga pKa that is greater than about 8.0 or less than about 6.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue selectedfrom the group consisting of His, Glu, Asp, Tyr, and Lys with anotheramino acid residue.

In some embodiments, at least one of the one or more amino acidsubstitutions are selected from the group consisting of: substitution ofa surface histidine with an arginine, substitution of a surface glutamicacid with a glutamine, and substitution of a surface lysine with anarginine.

In some embodiments, at least one of the one or more amino acidmodifications is a substitution of an amino acid residue with an alanineresidue.

In some embodiments of the method, at least one of the one or more aminoacid modifications in the polymerase is a deletion of an amino acid. Insome embodiments, at least one of the one or more deleted amino acids isan amino acid residue having a pKa of between about 4.0 and about 10.0.In some embodiments, the amino acid residue having a pKa that is greaterthan about 10.0 or less than about 4.0 is selected from the groupconsisting of: Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe,Ser, Thr, Trp, Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more deleted amino acidsis an amino acid residue having a pKa within the range of about 6.0 toabout 8.0 with another amino acid residue. In some embodiments, thedeleted amino acid is His, Glu, Asp, Tyr or Lys.

In some embodiments of the method, the polymerase comprises one or morechemical amino acid modifications that reduce the buffering capacity ofthe protein relative to the corresponding wild-type protein within therange of about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, orabout pH 7 to about pH 9. In some embodiments, the one or more chemicalamino acid modifications includes a chemical modification of theN-terminal amino acid. In some embodiments, the one or more chemicalamino acid modifications includes a chemical modification of an aminoacid residue including a primary amine group with an amine-reactiveagent. In further embodiments, the amine-reactive reagent includes anacylating agent or an activated ester.

In some embodiments, at least one of the one or more amino acidsubstitutions, deletions or chemical modifications includes asubstitution of an amino acid residue that is at least 20%, at least25%, at least 30%, at least 35% or at least 40% solvent exposed in thecorresponding wild-type protein with another amino acid residue.

It will be understood that the polymerase may comprise any combinationof amino acid substitutions, deletions or chemical modifications. Apolymerase comprising any one or more of such additions, substitutions,deletions and chemical modifications may be referred to a “modified”polymerase.

In some embodiments, the modified polymerase is selected from the groupconsisting of an A family DNA polymerase; a B family DNA polymerase; amixed-type polymerase; an unclassified DNA polymerase and RT familypolymerase; and variants and derivatives thereof.

In some embodiments, the DNA polymerase is an A family DNA polymeraseselected from the group consisting of a Pol I-type DNA polymerase suchas E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, Bst DNA polymerase, Taq DNA polymerase, Platinum Taq DNApolymerase series, T7 DNA polymerase, and Tth DNA polymerase. In someembodiments, the DNA polymerase is Bst DNA polymerase. In otherembodiments, the DNA polymerase is E. coli DNA polymerase. In someembodiments, the DNA polymerase is the Klenow fragment of E. coli DNApolymerase. In some embodiments, the polymerase is Taq DNA polymerase.In some embodiments, the polymerase is T7 DNA polymerase.

In other embodiments, the DNA polymerase is a B family DNA polymeraseselected from the group consisting of Tli polymerase, Pfu polymerase,Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase,Sac polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, Pho polymerase, ES4 polymerase, VENT polymerase, DEEPVENTpolymerase, phage Phi29 polymerase, and phage B103 polymerase. In someembodiments, the polymerase is phage Phi29 DNA polymerase. In someembodiments the polymerase is phage B103 polymerase, including, forexample, the variants disclosed in U.S. Patent Publication No.20110014612.

In other embodiments, the DNA polymerase is a mixed-type polymeraseselected from the group consisting of EX-Taq polymerase, LA-Taqpolymerase, Expand polymerase series, and Hi-Fi polymerase. In yet otherembodiments, the DNA polymerase is an unclassified DNA polymeraseselected from the group consisting of Tbr polymerase, Tfl polymerase,Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tihpolymerase, and Tfi polymerase.

In other embodiments, the DNA polymerase is an RT polymerase selectedfrom the group consisting of HIV reverse transcriptase, M-MLV reversetranscriptase and AMV reverse transcriptase. In some embodiments, thepolymerase is HIV reverse transcriptase or a fragment thereof having DNApolymerase activity.

In some embodiments, the DNA polymerase is a Bst DNA polymerasecomprising one or more amino acid substitutions that substantiallyreduce its buffering capacity within the range of about pH 4 to about pH10.

In some embodiments, the one or more amino acid substitutions in the BstDNA polymerase reduce the buffering capacity of the polymerase relativeto the corresponding wild-type (unsubstituted) polymerase within therange of about pH 7 to about pH 9.

In some embodiments, at least one of the one or more amino acidsubstitutions in the Bst DNA polymerase is a conservative amino acidsubstitution. In further embodiments, the at least one conservativeamino acid substitution is selected from the group consisting ofhistidine to arginine, glutamic acid to glutamine, aspartic acid toasparagine, lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, the one or more conservative amino acidsubstitutions are of one or more amino acid residues shown in Table 2.In some embodiments, the one or more conservative amino acidsubstitutions are selected from the group consisting of H46R, H273R,H281R, E446Q, H473R, H528R, H572R and Y477F, the numbering of amino acidresidues being in accordance with that of SEQ ID NO: 1.

In some embodiments, the one or more amino acid substitutions includes asubstitution of alanine at position 2 with Met, Asn, Gln, Leu, Ile, Phe,or Trp, the numbering of amino acid residues being in accordance withthat of SEQ ID NO:2.

In some embodiments, the Bst DNA polymerase comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:3 and SEQ ID NO: 4, or a variant thereof having one or more conservativeamino acid substitutions. In some embodiments, the Bst DNA polymerasecomprises the amino acid sequence of SEQ ID NO: 2. In some embodiments,the Bst DNA polymerase is a variant of a protein comprising the aminoacid sequence shown in SEQ ID NO: 2, wherein the variant comprises anamino acid sequence that is at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 2.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 3. In some embodiments, the Bst DNA polymerase isa variant of a protein comprising the amino acid sequence shown in SEQID NO: 3, wherein the variant comprises an amino acid sequence that isat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 3.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 4. In other embodiments, the Bst polymerase is avariant of a protein comprising the amino acid sequence shown in SEQ IDNO: 4, wherein the variant comprises an amino acid sequence that is atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 4.

In other embodiments of the method, the DNA polymerase is a Therminator™DNA polymerase comprising one or more conservative amino acidsubstitutions that substantially reduce its buffering capacity relativeto the corresponding wild-type protein within the range of about pH 7 toabout pH 9, wherein the one or more conservative amino acidsubstitutions are selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In other embodiments of the method, the DNA polymerase is a KOD DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 6.

In other embodiments of the method, the DNA polymerase is a B103 DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 7.

In some embodiments, the method for obtaining sequence information froma nucleic acid template further comprises providing an SSB, wherein theSSB comprises one or more amino acid substitutions that substantiallyreduce its buffering capacity within the range of about pH 4 to about pH10.

In some embodiments, the one or more amino acid substitutions in the SSBreduce the buffering capacity of the SSB relative to the correspondingwild-type SSB within the range of about pH 7 to about pH 9.

In some embodiments, at least one of the one or more amino acidsubstitutions in the SSB is a conservative amino acid substitution. Infurther embodiments, the at least one conservative amino acidsubstitution is selected from the group consisting of histidine toarginine, glutamic acid to glutamine, aspartic acid to asparagine,lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, the SSB is E. coli SSB. In further embodiments, theone or more conservative amino acid substitutions are of one or moreamino acid residues shown in Table 2. In some embodiments, the SSBcomprises the amino acid substitution K7R.

In some embodiments, the disclosure relates generally to a method forsequencing a nucleic acid, comprising: disposing a plurality of templatenucleic acids into a plurality of reaction chambers, wherein one or moreof the reaction chambers are in contact with a field effect transistor(FET) array, and contacting at least one of the template nucleic acidswith a polymerase including one or more conservative amino acidsubstitutions that substantially reduce its buffering capacity withinthe range of pH 7 to pH 9; synthesizing a new nucleic acid strand bysequentially incorporating one or more nucleotides into a nucleic acidmolecule and generating one or more hydrogen ions as a byproduct of suchnucleotide incorporation; and detecting the incorporation of the one ormore nucleotides by detecting the generation of the one or more hydrogenions using the FET.

In some embodiments, the detecting includes detecting a change involtage and/or current at the at least one FET within the array inresponse to the generation of the one or more hydrogen ions.

In some embodiments, the FET is selected from the group consisting of:ion-sensitive FET (isFET) and chemically-sensitive FET (chemFET).

In some embodiments, the polymerase is Bst DNA polymerase. In furtherembodiments, the one or more conservative amino acid substitutionswithin the polymerase are of one or more amino acid residues shown inTable 2 or Table 3. In further embodiments, the one or more conservativeamino acid substitutions are selected from the group consisting ofH341R, H568R, H576R, E741Q, H768R, H823R, H867R and Y772F. In someembodiments, the polymerase is selected from the group consisting of SEQID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a variant thereof having oneor more conservative amino acid substitutions.

In some embodiments, the method further comprises providing an SSB,wherein the SSB comprises one or more conservative amino acidsubstitutions that substantially remove its buffering capacity withinthe range of pH 7 to pH 9. In further embodiments, the SSB is E. coliSSB. In further embodiments, the one or more conservative amino acidsubstitutions in the SSB are of one or more amino acid residues shown inTable 4.

In another embodiment, the disclosure relates generally to a method forobtaining sequence information from a nucleic acid comprising:

(a) disposing a plurality of solid or semi-solid supports into aplurality of reaction chambers on a sensor array formed in asemiconductor substrate, at least one reaction chamber including apolymerase, a sequencing primer and a single solid or semi-solid supportattached to a plurality of template nucleic acids having at least 80%sequence identity to each other, wherein the polymerase comprises one ormore amino acid substitutions that substantially remove its bufferingcapacity within the range of pH 7 to pH 9, and each reaction chamber isin contact with or capacitively coupled to a sensor including a FETconfigured to provide at least one output representing the presence ofone or more hydrogen ions in the reaction chamber;

(b) introducing at least one nucleotide into the at least one reactionchamber and incorporating the at least one nucleotide into the primerusing the polymerase, thereby generating one or more hydrogen ionbyproducts;

(c) detecting the incorporation of the at least one nucleotides bydetecting the presence of the hydrogen ion byproducts.

In some embodiments, the method further includes repeating steps (a)through (c) until the nucleic acid is sequenced.

In some embodiments, the method further includes a step of washingunincorporated nucleotides from the at least one reaction chambers.

In some embodiments, the method further includes repeating steps (a)through (c) as well as the washing step until the nucleic acid issequenced.

In some embodiments, the polymerase is Bst DNA polymerase. In furtherembodiments, the one or more conservative amino acid substitutionswithin the polymerase are of one or more amino acid residues shown inTable 2 or Table 3. In further embodiments, the one or more conservativeamino acid substitutions are selected from the group consisting ofH341R, H568R, H576R, E741Q, H768R, H823R, H867R and Y772F. In someembodiments, the polymerase is selected from the group consisting of SEQID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a variant thereof having oneor more conservative amino acid substitutions.

In some embodiments, the method further comprises providing an SSB,wherein the SSB comprises one or more conservative amino acidsubstitutions that substantially remove its buffering capacity withinthe range of pH 7 to pH 9. In further embodiments, the SSB is E. coliSSB. In further embodiments, the one or more conservative amino acidsubstitutions in the SSB are of one or more amino acid residues shown inTable 4.

In a further embodiment, the disclosure relates generally to a methodfor reducing the buffering capacity of a protein used in a DNAsequencing or amplification reaction, comprising making one or moreamino acid substitutions in the protein sequence that substantiallyreduce the protein's buffering capacity within the range of about pH 4to about pH 10.

In some embodiments, the amino acid substitutions substantially reducethe protein's buffering capacity within the range of about pH 7 to aboutpH 9.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa in the range of about 4 to about 10 with an amino acid residuehaving a pKa less than about 4 or greater than about 10.

In some embodiments, the protein is a DNA- or RNA-binding protein.

In various embodiments, the protein is a DNA polymerase selected fromthe group consisting of an A family DNA polymerase; a B familypolymerase; a mixed-type polymerase; an unclassified DNA polymerase; anRT family polymerase; and variants and derivatives thereof.

In some embodiments, the DNA polymerase is an A family DNA polymeraseselected from the group consisting of a Pol I-type DNA polymerase suchas E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, Bst DNA polymerase, Taq DNA polymerase, Platinum Taq DNApolymerase series, T7 DNA polymerase, and Tth DNA polymerase; a B familyDNA polymerase selected from the group consisting of, Tli polymerase,Pfu polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase,KOD polymerase, Sac polymerase, Sso polymerase, Poc polymerase, Pabpolymerase, Mth polymerase, Pho polymerase, ES4 polymerase, VENTpolymerase, DEEPVENT polymerase, phage Phi29 polymerase, and phage B103polymerase; a mixed-type polymerase selected from the group consistingof EX-Taq polymerase, LA-Taq polymerase, Expand polymerase series, andHi-Fi polymerase; an unclassified DNA polymerase selected from the groupconsisting of Tbr polymerase, Tfl polymerase, Tru polymerase, Tacpolymerase, Tne polymerase, Tma polymerase, Tih polymerase, and Tfipolymerase; and an RT polymerase selected from the group consisting ofHIV reverse transcriptase, M-MLV reverse transcriptase and AMV reversetranscriptase.

In some embodiments, the polymerase is Bst DNA polymerase. In furtherembodiments, the one or more conservative amino acid substitutionswithin the polymerase are of one or more amino acid residues shown inTable 2 or Table 3. In further embodiments, the one or more conservativeamino acid substitutions are selected from the group consisting ofH341R, H568R, H576R, E741Q, H768R, H823R, H867R and Y772F. In someembodiments, the polymerase is selected from the group consisting of SEQID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a variant thereof having oneor more conservative amino acid substitutions.

In some embodiments, the method further comprises providing an SSB,wherein the SSB comprises one or more conservative amino acidsubstitutions that substantially remove its buffering capacity withinthe range of pH 7 to pH 9. In further embodiments, the SSB is E. coliSSB. In further embodiments, the one or more conservative amino acidsubstitutions in the SSB are of one or more amino acid residues shown inTable 2.

In a further embodiment, the disclosure relates generally to a method ofdetecting a nucleotide incorporation, comprising:

(a) performing a nucleotide incorporation using a modified polymeraseand generating one or more hydrogen ions as a by-product of thenucleotide incorporation, where the modified polymerase includes one ormore amino acid substitutions that reduce the buffering capacity of themodified polymerase relative to the unmodified polymerase; and

(b) detecting the presence of the one or more hydrogen ions generated asa by-product of the nucleotide incorporation, thereby detecting thenucleotide incorporation.

In various embodiments, the polymerase may be any of the modifiedpolymerases containing any one or more of the substitutions, deletionsor chemical modifications described herein.

In some embodiments, the modified polymerase includes one or more aminoacid substitutions that reduce the buffering capacity of the modifiedpolymerase within the pH range of about 4 to about 10 relative to theunmodified polymerase.

In some embodiments, the one or more amino acid substitutions in thepolymerase reduce the buffering capacity of the polymerase relative tothe corresponding wild-type (unsubstituted) polymerase within the rangeof about pH 7 to about pH 9.

In some embodiments, at least one of the one or more amino acidsubstitutions in the polymerase is a conservative amino acidsubstitution. In further embodiments, the at least one conservativeamino acid substitution is selected from the group consisting ofhistidine to arginine, glutamic acid to glutamine, aspartic acid toasparagine, lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, the polymerase comprises one or more amino acidsubstitutions that reduce the buffering capacity of said polymeraserelative to the corresponding wild-type polymerase within the range ofabout pH 4 to about pH 10, wherein at least one of the one or more aminoacid substitutions includes a substitution of an amino acid residuehaving a pKa within the range of about 4.0 to about 10.0 with anotheramino acid residue. In some embodiments, the pKa of the amino acidresidue is a solution pKa of the amino acid residue. In otherembodiments, the pKa of the amino acid residue is a pKa of the aminoacid residue in the context of the corresponding wild-type protein.

In some embodiments, at least one of the one or more amino substitutionsincludes a substitution of an amino acid residue having a pKa of betweenabout 4.0 and about 10.0 with an amino acid residue having a pKa that isgreater than about 10.0 or less than about 4.0. In further embodimentsthe amino acid residue having a pKa that is greater than about 10.0 orless than about 4.0 is selected from the group consisting of: Arg, Asp,Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp, Val andN-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 6.0 to about 8.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidmodifications includes a substitution of an amino acid residue having apKa of between about 6.0 and about 8.0 with an amino acid residue havinga pKa that is greater than about 8.0 or less than about 6.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue selectedfrom the group consisting of His, Glu, Asp, Tyr, and Lys with anotheramino acid residue.

In some embodiments, at least one of the one or more amino acidmodifications is a substitution of an amino acid residue with an alanineresidue.

In some embodiments of the method, at least one of the one or more aminoacid modifications in the polymerase is a deletion of an amino acid. Insome embodiments, at least one of the one or more deleted amino acids isan amino acid residue having a pKa of between about 4.0 and about 10.0.In some embodiments, the amino acid residue having a pKa that is greaterthan about 10.0 or less than about 4.0 is selected from the groupconsisting of: Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe,Ser, Thr, Trp, Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more deleted amino acidsis an amino acid residue having a pKa within the range of about 6.0 toabout 8.0 with another amino acid residue. In some embodiments, thedeleted amino acid is His, Glu, Asp, Tyr or Lys.

In some embodiments of the method, the polymerase comprises one or morechemical amino acid modifications that reduce the buffering capacity ofthe protein relative to the corresponding wild-type protein within therange of about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, orabout pH 7 to about pH 9. In some embodiments, the one or more chemicalamino acid modifications includes a chemical modification of theN-terminal amino acid. In some embodiments, the one or more chemicalamino acid modifications includes a chemical modification of an aminoacid residue including a primary amine group with an amine-reactiveagent. In further embodiments, the amine-reactive reagent includes anacylating agent or an activated ester.

In some embodiments, at least one of the one or more amino acidsubstitutions, deletions or chemical modifications includes asubstitution of an amino acid residue that is at least 20%, at least25%, at least 30%, at least 35% or at least 40% solvent exposed in thecorresponding wild-type protein with another amino acid residue.

In some embodiments, the modified polymerase is selected from the groupconsisting of an A family DNA polymerase; a B family DNA polymerase; amixed-type polymerase; an unclassified DNA polymerase and RT familypolymerase; and variants and derivatives thereof.

In some embodiments, the DNA polymerase is an A family DNA polymeraseselected from the group consisting of a Pol I-type DNA polymerase suchas E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, Bst DNA polymerase, Taq DNA polymerase, Platinum Taq DNApolymerase series, T7 DNA polymerase, and Tth DNA polymerase. In someembodiments, the DNA polymerase is Bst DNA polymerase. In otherembodiments, the DNA polymerase is E. coli DNA polymerase. In someembodiments, the DNA polymerase is the Klenow fragment of E. coli DNApolymerase. In some embodiments, the polymerase is Taq DNA polymerase.In some embodiments, the polymerase is T7 DNA polymerase.

In other embodiments, the DNA polymerase is a B family DNA polymeraseselected from the group consisting of Tli polymerase, Pfu polymerase,Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase,Sac polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, Pho polymerase, ES4 polymerase, VENT polymerase, DEEPVENTpolymerase, phage Phi29 polymerase, and phage B103 polymerase. In someembodiments, the polymerase is phage Phi29 DNA polymerase. In someembodiments the polymerase is phage B103 polymerase, including, forexample, the variants disclosed in U.S. Patent Publication No.20110014612.

In other embodiments, the DNA polymerase is a mixed-type polymeraseselected from the group consisting of EX-Taq polymerase, LA-Taqpolymerase, Expand polymerase series, and Hi-Fi polymerase. In yet otherembodiments, the DNA polymerase is an unclassified DNA polymeraseselected from the group consisting of Tbr polymerase, Tfl polymerase,Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tihpolymerase, and Tfi polymerase.

In other embodiments, the DNA polymerase is an RT polymerase selectedfrom the group consisting of HIV reverse transcriptase, M-MLV reversetranscriptase and AMV reverse transcriptase. In some embodiments, thepolymerase is HIV reverse transcriptase or a fragment thereof having DNApolymerase activity.

In some embodiments, the DNA polymerase is a Bst DNA polymerasecomprising one or more amino acid substitutions that substantiallyreduce its buffering capacity within the range of about pH 4 to about pH10.

In some embodiments, the one or more amino acid substitutions in the BstDNA polymerase reduce the buffering capacity of the polymerase relativeto the corresponding wild-type (unsubstituted) polymerase within therange of about pH 7 to about pH 9.

In some embodiments, at least one of the one or more amino acidsubstitutions in the Bst DNA polymerase is a conservative amino acidsubstitution. In further embodiments, the at least one conservativeamino acid substitution is selected from the group consisting ofhistidine to arginine, glutamic acid to glutamine, aspartic acid toasparagine, lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, the one or more conservative amino acidsubstitutions are of one or more amino acid residues shown in Table 2.In some embodiments, the one or more conservative amino acidsubstitutions are selected from the group consisting of H46R, H273R,H281R, E446Q, H473R, H528R, H572R and Y477F, the numbering of amino acidresidues being in accordance with that of SEQ ID NO: 1.

In some embodiments, the one or more amino acid substitutions includes asubstitution of alanine at position 2 with Met, Asn, Gln, Leu, Ile, Phe,or Trp, the numbering of amino acid residues being in accordance withthat of SEQ ID NO:2.

In some embodiments, the Bst DNA polymerase comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:3 and SEQ ID NO: 4, or a variant thereof having one or more conservativeamino acid substitutions. In some embodiments, the Bst DNA polymerasecomprises the amino acid sequence of SEQ ID NO: 2. In some embodiments,the Bst DNA polymerase is a variant of a protein comprising the aminoacid sequence shown in SEQ ID NO: 2, wherein the variant comprises anamino acid sequence that is at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 2.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 3. In some embodiments, the Bst DNA polymerase isa variant of a protein comprising the amino acid sequence shown in SEQID NO: 3, wherein the variant comprises an amino acid sequence that isat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 3.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 4. In other embodiments, the Bst polymerase is avariant of a protein comprising the amino acid sequence shown in SEQ IDNO: 4, wherein the variant comprises an amino acid sequence that is atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 4.

In other embodiments of the method, the DNA polymerase is a Therminator™DNA polymerase comprising one or more conservative amino acidsubstitutions that substantially reduce its buffering capacity relativeto the corresponding wild-type protein within the range of about pH 7 toabout pH 9, wherein the one or more conservative amino acidsubstitutions are selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In other embodiments of the method, the DNA polymerase is a KOD DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 6.

In other embodiments of the method, the DNA polymerase is a B103 DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially reduce its buffering capacity relative to thecorresponding wild-type protein within the range of about pH 7 to aboutpH 9, wherein the one or more conservative amino acid substitutions areselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 7.

In some embodiments, the method further comprises providing an SSB,wherein the SSB comprises one or more amino acid substitutions thatsubstantially reduce its buffering capacity within the range of about pH4 to about pH 10.

In some embodiments, the one or more amino acid substitutions in the SSBreduce the buffering capacity of the SSB relative to the correspondingwild-type SSB within the range of about pH 7 to about pH 9.

In some embodiments, at least one of the one or more amino acidsubstitutions in the SSB is a conservative amino acid substitution. Infurther embodiments, the at least one conservative amino acidsubstitution is selected from the group consisting of histidine toarginine, glutamic acid to glutamine, aspartic acid to asparagine,lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, the SSB is E. coli SSB. In further embodiments, theone or more conservative amino acid substitutions are of one or moreamino acid residues shown in Table 2. In some embodiments, the SSBcomprises the amino acid substitution K7R.

In some embodiments of the method of detecting a nucleotideincorporation, the detecting comprises using an ion-sensitive fieldeffect transistor (ISFET). In some embodiments the ISFET is in an ISFETarray.

In some embodiments the reaction is performed in a reaction chambercomprising or capacitively coupled to a chemFET.

In some embodiments, the reaction is in the presence of an agent thatalters the value of a pKa of at least an amino acid in the modifiedpolypeptide from within the range of about 6 to about 8 to less thanabout 6 or greater than about 8. In some embodiments, the agent is aphospholipid, a sulfonic acid surfactant, a polyanionic electrolyte or asalt thereof, a polycationic electrolyte or a salt thereof, tetramethylammonium or a salt thereof, or a combination thereof.

In some embodiments, the method further comprises repeating steps a)-b).

In some embodiments, the disclosure relates generally to methods forsequencing a nucleic acid, comprising: providing a template nucleic acidhybridized to a sequencing primer and bound to a polymerase;synthesizing a new nucleic acid strand by incorporating one or moreknown nucleoside triphosphates sequentially at the 3′ end of thesequencing primer; and detecting such incorporation at the 3′ end of theprimer by measuring a concentration of a hydrogen ion byproductgenerated if the known nucleoside triphosphate is complementary tocorresponding nucleotides in the template nucleic acid.

In some embodiments, the one or more amino acid substitutions in thepolymerase may include any one or more of the amino acid substitutionsdescribed herein.

In some embodiments, at least one of the one or more amino acidsubstitutions can be a conservative amino acid substitution.

In some embodiments, each of the one or more amino acid substitutions isa conservative amino acid substitution.

In some embodiments, the polymerase includes any one of the modifiedpolymerases described herein. In some embodiments, the polymerase is abufferless polymerase. For example, the polymerase can have reducedbuffering capacity relative to the corresponding unsubstitutedpolymerase.

In some embodiments, the polymerase includes one or more amino acidsubstitutions that substantially remove the buffering capacity of thepolymerase within the pH range of about 4 to about 10 relative to thecorresponding unsubstituted polymerase. The unsubstituted polymerase canbe the wild-type version of the polymerase.

In some embodiments, the one or more amino acid substitutions in thepolymerase substantially remove the buffering capacity of the polymeraserelative to the corresponding unsubstituted polymerase within the rangeof about pH 7 to about pH 9. The unsubstituted polymerase can be thewild-type version of the polymerase.

In some embodiments, the polymerase includes one or more amino acidsubstitutions that substantially reduce the buffering capacity of thepolymerase within the pH range of about 4 to about 10 relative to thecorresponding unsubstituted polymerase. The unsubstituted polymerase canbe the wild-type version of the polymerase.

In some embodiments, the one or more amino acid substitutions in thepolymerase substantially reduce the buffering capacity of the polymeraserelative to the corresponding unsubstituted polymerase within the rangeof about pH 7 to about pH 9. The unsubstituted polymerase can be thewild-type version of the polymerase.

In some embodiments, at least one of the one or more amino acidsubstitutions in the polymerase is a conservative amino acidsubstitution that is selected from the group consisting of histidine toarginine, glutamic acid to glutamine, aspartic acid to asparagine,lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 4.0 to about 10.0 with another amino acidresidue. In some embodiments, the pKa of the amino acid residue is asolution pKa of the amino acid residue. In other embodiments, the pKa ofthe amino acid residue is a pKa of the amino acid residue in the contextof the corresponding wild-type protein.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 7 to about 9 with another amino acidresidue. In some embodiments, the pKa of the amino acid residue is asolution pKa of the amino acid residue. In other embodiments, the pKa ofthe amino acid residue is a pKa of the amino acid residue in the contextof the corresponding wild-type protein.

In some embodiments, at least one of the one or more conservative aminosubstitutions includes a substitution of an amino acid residue having apKa of between about 4.0 and about 10.0 with an amino acid residuehaving a pKa that is greater than about 10.0 or less than about 4.0. Infurther embodiments the amino acid residue having a pKa that is greaterthan about 10.0 or less than about 4.0 is selected from the groupconsisting of: Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe,Ser, Thr, Trp, Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more conservative aminosubstitutions includes a substitution of an amino acid residue having apKa of between about 7 and about 9 with an amino acid residue having apKa that is greater than about 9 or less than about 7. In furtherembodiments the amino acid residue having a pKa that is greater thanabout 9 or less than about 7 is selected from the group consisting of:Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp,Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 6.0 to about 8.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa of between about 6.0 and about 8.0 with an amino acid residue havinga pKa that is greater than about 8.0 or less than about 6.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 7.0 to about 9.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa of between about 7.0 and about 9.0 with an amino acid residue havinga pKa that is greater than about 9.0 or less than about 7.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue selectedfrom the group consisting of His, Glu, Asp, Tyr, and Lys with anotheramino acid residue.

In some embodiments, at least one of the one or more amino acidsubstitutions is a substitution of an amino acid residue with an alanineresidue.

In some embodiments, the polymerase comprises one or more conservativeamino acid substitutions that reduce the buffering capacity of theprotein relative to the corresponding wild-type protein within the rangeof about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, or about pH7 to about pH 9. In some embodiments, at least one of the one or moreamino acid substitutions includes a substitution of an amino acidresidue that is at least 20%, at least 25%, at least 30%, at least 35%or at least 40% solvent exposed in the corresponding wild-type proteinwith another amino acid residue.

In some embodiments, at least one of the one or more amino acidsubstitutions is a substitution of an amino acid residue with an alanineresidue.

In some embodiments, the polymerase comprises one or more amino acidconservative amino acid substitutions that reduce the buffering capacityof the protein relative to the corresponding wild-type protein withinthe range of about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, orabout pH 7 to about pH 9. In some embodiments, at least one of the oneor more amino acid substitutions includes a substitution of an aminoacid residue that is at least 20%, at least 25%, at least 30%, at least35% or at least 40% solvent exposed in the corresponding wild-typeprotein with another amino acid residue.

In some embodiments, the polymerase comprises one or more conservativeamino acid substitutions that substantially remove the bufferingcapacity of the polymerase within the range of about pH 4 to about pH10, about pH 5.5 to about pH 9.5, or about pH 7 to about pH 9.

In some embodiments, the polymerase is selected from the groupconsisting of an A family DNA polymerase; a B family DNA polymerase; amixed-type polymerase; an unclassified DNA polymerase and RT familypolymerase; and variants and derivatives thereof.

In some embodiments, the DNA polymerase is an A family DNA polymeraseselected from the group consisting of a Pol I-type DNA polymerase suchas E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, Bst DNA polymerase, Taq DNA polymerase, Platinum Taq DNApolymerase series, T7 DNA polymerase, and Tth DNA polymerase. In someembodiments, the DNA polymerase is Bst DNA polymerase. In otherembodiments, the DNA polymerase is E. coli DNA polymerase. In someembodiments, the DNA polymerase is the Klenow fragment of E. coli DNApolymerase. In some embodiments, the polymerase is Taq DNA polymerase.In some embodiments, the polymerase is T7 DNA polymerase.

In other embodiments, the DNA polymerase is a B family DNA polymeraseselected from the group consisting of Tli polymerase, Pfu polymerase,Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase,Sac polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, Pho polymerase, ES4 polymerase, VENT polymerase, DEEPVENTpolymerase, phage Phi29 polymerase, and phage B103 polymerase. In someembodiments, the polymerase is phage Phi29 DNA polymerase. In someembodiments the polymerase is phage B103 polymerase, including, forexample, the variants disclosed in U.S. Patent Publication No.20110014612.

In other embodiments, the DNA polymerase is a mixed-type polymeraseselected from the group consisting of EX-Taq polymerase, LA-Taqpolymerase, Expand polymerase series, and Hi-Fi polymerase. In yet otherembodiments, the DNA polymerase is an unclassified DNA polymeraseselected from the group consisting of Tbr polymerase, Tfl polymerase,Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tihpolymerase, and Tfi polymerase.

In other embodiments, the DNA polymerase is an RT polymerase selectedfrom the group consisting of HIV reverse transcriptase, M-MLV reversetranscriptase and AMV reverse transcriptase. In some embodiments, thepolymerase is HIV reverse transcriptase or a fragment thereof having DNApolymerase activity.

In some embodiments, the DNA polymerase is a Bst DNA polymerasecomprising one or more amino acid substitutions that substantiallyreduce its buffering capacity within the range of about pH 4 to about pH10. In some embodiments, the one or more amino acid substitutionssubstantially remove the buffering capacity of the polymerase within therange of about pH 4 to about pH 10.

In some embodiments, the one or more amino acid substitutions in the BstDNA polymerase substantially reduce the buffering capacity of thepolymerase relative to the corresponding unsubstituted polymerase withinthe range of about pH 7 to about pH 9. In some embodiments, the one ormore amino acid substitutions substantially remove the bufferingcapacity of the Bst polymerase within the range of about pH 7 to aboutpH 9. In some embodiments, the one or more amino acid substitutionssubstantially reduce the buffering capacity of the Bst polymeraserelative to the corresponding unsubstituted Bst polymerase within therange of about pH 7 to about pH 9. In some embodiments, theunsubstituted polymerase can be the wild-type version of the Bstpolymerase.

In some embodiments, at least one of the one or more amino acidsubstitutions in the Bst DNA polymerase is a conservative amino acidsubstitution. In further embodiments, the at least one conservativeamino acid substitution is selected from the group consisting ofhistidine to arginine, glutamic acid to glutamine, aspartic acid toasparagine, lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, the one or more conservative amino acidsubstitutions are of one or more amino acid residues shown in Table 2.In some embodiments, the one or more conservative amino acidsubstitutions are selected from the group consisting of H46R, H273R,H281R, E446Q, H473R, H528R, H572R and Y477F, the numbering of amino acidresidues being in accordance with that of SEQ ID NO: 1.

In some embodiments, the one or more amino acid substitutions includes asubstitution of alanine at position 2 with Met, Asn, Gln, Leu, Ile, Phe,or Trp, the numbering of amino acid residues being in accordance withthat of SEQ ID NO:2.

In some embodiments, the Bst DNA polymerase comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:3 and SEQ ID NO: 4, or a variant thereof having one or more conservativeamino acid substitutions. In some embodiments, the Bst DNA polymerasecomprises the amino acid sequence of SEQ ID NO: 2. In some embodiments,the Bst DNA polymerase is a variant of a protein comprising the aminoacid sequence shown in SEQ ID NO: 2, wherein the variant comprises anamino acid sequence that is at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 2.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 3. In some embodiments, the Bst DNA polymerase isa variant of a protein comprising the amino acid sequence shown in SEQID NO: 3, wherein the variant comprises an amino acid sequence that isat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 3.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 4. In other embodiments, the Bst polymerase is avariant of a protein comprising the amino acid sequence shown in SEQ IDNO: 4, wherein the variant comprises an amino acid sequence that is atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 4.

In some embodiments, the DNA polymerase is a Therminator™ DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In some embodiments, the DNA polymerase is a Therminator™ DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially remove its buffering capacity within the range of about pH7 to about pH 9, wherein the one or more conservative amino acidsubstitutions are selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In some embodiments, the DNA polymerase is a KOD DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table6.

In some embodiments, the DNA polymerase is a KOD DNA polymerasecomprising one or more amino acid substitutions that substantiallyremove its buffering capacity within the range of about pH 7 to about pH9. The one or more conservative amino acid substitutions are optionallyselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 6.

In some embodiments, the DNA polymerase is a B103 DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table7.

In some embodiments, the DNA polymerase is a B103 DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 4 toabout pH 10. The unsubstituted polymerase can be the wild-type versionof the polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table7.

In other embodiments of the method, the DNA polymerase is a B103 DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially remove its buffering capacity within the range ofabout pH 7 to about pH 9. The one or more conservative amino acidsubstitutions are optionally selected from the group consisting of:histidine to arginine, glutamic acid to glutamine, lysine to arginineand tyrosine to phenylalanine. In some embodiments, the one or moreconservative amino acid substitutions are of one or more amino acidresidues shown in Table 7.

In some embodiments, the disclosure relates generally to methods forsequencing a nucleic acid, comprising: (a) disposing a plurality oftemplate nucleic acids into a plurality of reaction chambers, whereinthe plurality of reaction chambers is in contact with achemical-sensitive field effect transistor (chemFET) array, the chemFETarray comprising at least one chemFET for each reaction chamber, andwherein each of the template nucleic acids is bound to a polymerase,wherein the polymerase comprises one or more amino acid modificationsthat substantially reduce its buffering capacity relative to thecorresponding unmodified polymerase; (b) synthesizing a new nucleic acidstrand by sequentially incorporating one or more known nucleosidetriphosphates at the 3′ end of the sequencing primer, wherein a hydrogenion byproduct is generated when the known nucleoside triphosphate iscomplementary to corresponding nucleotides in the template nucleic acid;and (c) detecting the incorporation of the one or more known nucleosidetriphosphates by a change in voltage and/or current at the at least onechemFET within the array in response to a change in hydrogen ionconcentration proximate thereto.

In some embodiments, the one or more amino acid substitutions in thepolymerase may include any one or more of the amino acid substitutionsdescribed herein.

In some embodiments, at least one of the one or more amino acidsubstitutions can be a conservative amino acid substitution.

In some embodiments, each of the one or more amino acid substitutions isa conservative amino acid substitution.

In some embodiments, the polymerase includes any one of the modifiedpolymerases described herein. In some embodiments, the polymerase is abufferless polymerase. For example, the polymerase can have reducedbuffering capacity relative to the corresponding unsubstitutedpolymerase.

In some embodiments, the polymerase includes one or more amino acidsubstitutions that substantially remove the buffering capacity of thepolymerase within the pH range of about 4 to about 10 relative to thecorresponding unsubstituted polymerase. The unsubstituted polymerase canbe the wild-type version of the polymerase.

In some embodiments, the one or more amino acid substitutions in thepolymerase substantially remove the buffering capacity of the polymeraserelative to the corresponding unsubstituted polymerase within the rangeof about pH 7 to about pH 9. The unsubstituted polymerase can be thewild-type version of the polymerase.

In some embodiments, the polymerase includes one or more amino acidsubstitutions that substantially reduce the buffering capacity of thepolymerase within the pH range of about 4 to about 10 relative to thecorresponding unsubstituted polymerase. The unsubstituted polymerase canbe the wild-type version of the polymerase.

In some embodiments, the one or more amino acid substitutions in thepolymerase substantially reduce the buffering capacity of the polymeraserelative to the corresponding unsubstituted polymerase within the rangeof about pH 7 to about pH 9. The unsubstituted polymerase can be thewild-type version of the polymerase.

In some embodiments, at least one of the one or more amino acidsubstitutions in the polymerase is a conservative amino acidsubstitution that is selected from the group consisting of histidine toarginine, glutamic acid to glutamine, aspartic acid to asparagine,lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 4.0 to about 10.0 with another amino acidresidue. In some embodiments, the pKa of the amino acid residue is asolution pKa of the amino acid residue. In other embodiments, the pKa ofthe amino acid residue is a pKa of the amino acid residue in the contextof the corresponding wild-type protein.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 7 to about 9 with another amino acidresidue. In some embodiments, the pKa of the amino acid residue is asolution pKa of the amino acid residue. In other embodiments, the pKa ofthe amino acid residue is a pKa of the amino acid residue in the contextof the corresponding wild-type protein.

In some embodiments, at least one of the one or more conservative aminosubstitutions includes a substitution of an amino acid residue having apKa of between about 4.0 and about 10.0 with an amino acid residuehaving a pKa that is greater than about 10.0 or less than about 4.0. Infurther embodiments the amino acid residue having a pKa that is greaterthan about 10.0 or less than about 4.0 is selected from the groupconsisting of: Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe,Ser, Thr, Trp, Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more conservative aminosubstitutions includes a substitution of an amino acid residue having apKa of between about 7 and about 9 with an amino acid residue having apKa that is greater than about 9 or less than about 7. In furtherembodiments the amino acid residue having a pKa that is greater thanabout 9 or less than about 7 is selected from the group consisting of:Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp,Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 6.0 to about 8.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa of between about 6.0 and about 8.0 with an amino acid residue havinga pKa that is greater than about 8.0 or less than about 6.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 7.0 to about 9.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa of between about 7.0 and about 9.0 with an amino acid residue havinga pKa that is greater than about 9.0 or less than about 7.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue selectedfrom the group consisting of His, Glu, Asp, Tyr, and Lys with anotheramino acid residue.

In some embodiments, at least one of the one or more amino acidsubstitutions is a substitution of an amino acid residue with an alanineresidue.

In some embodiments, the polymerase comprises one or more conservativeamino acid substitutions that reduce the buffering capacity of theprotein relative to the corresponding wild-type protein within the rangeof about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, or about pH7 to about pH 9. In some embodiments, at least one of the one or moreamino acid substitutions includes a substitution of an amino acidresidue that is at least 20%, at least 25%, at least 30%, at least 35%or at least 40% solvent exposed in the corresponding wild-type proteinwith another amino acid residue.

In some embodiments, at least one of the one or more amino acidsubstitutions is a substitution of an amino acid residue with an alanineresidue.

In some embodiments, the polymerase comprises one or more amino acidconservative amino acid substitutions that reduce the buffering capacityof the protein relative to the corresponding wild-type protein withinthe range of about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, orabout pH 7 to about pH 9. In some embodiments, at least one of the oneor more amino acid substitutions includes a substitution of an aminoacid residue that is at least 20%, at least 25%, at least 30%, at least35% or at least 40% solvent exposed in the corresponding wild-typeprotein with another amino acid residue.

In some embodiments, the polymerase comprises one or more conservativeamino acid substitutions that substantially remove the bufferingcapacity of the polymerase within the range of about pH 4 to about pH10, about pH 5.5 to about pH 9.5, or about pH 7 to about pH 9.

In some embodiments, the polymerase is selected from the groupconsisting of an A family DNA polymerase; a B family DNA polymerase; amixed-type polymerase; an unclassified DNA polymerase and RT familypolymerase; and variants and derivatives thereof.

In some embodiments, the DNA polymerase is an A family DNA polymeraseselected from the group consisting of a Pol I-type DNA polymerase suchas E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, Bst DNA polymerase, Taq DNA polymerase, Platinum Taq DNApolymerase series, T7 DNA polymerase, and Tth DNA polymerase. In someembodiments, the DNA polymerase is Bst DNA polymerase. In otherembodiments, the DNA polymerase is E. coli DNA polymerase. In someembodiments, the DNA polymerase is the Klenow fragment of E. coli DNApolymerase. In some embodiments, the polymerase is Taq DNA polymerase.In some embodiments, the polymerase is T7 DNA polymerase.

In other embodiments, the DNA polymerase is a B family DNA polymeraseselected from the group consisting of Tli polymerase, Pfu polymerase,Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase,Sac polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, Pho polymerase, ES4 polymerase, VENT polymerase, DEEPVENTpolymerase, phage Phi29 polymerase, and phage B103 polymerase. In someembodiments, the polymerase is phage Phi29 DNA polymerase. In someembodiments the polymerase is phage B103 polymerase, including, forexample, the variants disclosed in U.S. Patent Publication No.20110014612.

In other embodiments, the DNA polymerase is a mixed-type polymeraseselected from the group consisting of EX-Taq polymerase, LA-Taqpolymerase, Expand polymerase series, and Hi-Fi polymerase. In yet otherembodiments, the DNA polymerase is an unclassified DNA polymeraseselected from the group consisting of Tbr polymerase, Tfl polymerase,Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tihpolymerase, and Tfi polymerase.

In other embodiments, the DNA polymerase is an RT polymerase selectedfrom the group consisting of HIV reverse transcriptase, M-MLV reversetranscriptase and AMV reverse transcriptase. In some embodiments, thepolymerase is HIV reverse transcriptase or a fragment thereof having DNApolymerase activity.

In some embodiments, the DNA polymerase is a Bst DNA polymerasecomprising one or more amino acid substitutions that substantiallyreduce its buffering capacity within the range of about pH 4 to about pH10. In some embodiments, the one or more amino acid substitutionssubstantially remove the buffering capacity of the polymerase within therange of about pH 4 to about pH 10.

In some embodiments, the one or more amino acid substitutions in the BstDNA polymerase substantially reduce the buffering capacity of thepolymerase relative to the corresponding unsubstituted polymerase withinthe range of about pH 7 to about pH 9. In some embodiments, the one ormore amino acid substitutions substantially remove the bufferingcapacity of the Bst polymerase within the range of about pH 7 to aboutpH 9. In some embodiments, the one or more amino acid substitutionssubstantially reduce the buffering capacity of the Bst polymeraserelative to the corresponding unsubstituted Bst polymerase within therange of about pH 7 to about pH 9. In some embodiments, theunsubstituted polymerase can be the wild-type version of the Bstpolymerase.

In some embodiments, at least one of the one or more amino acidsubstitutions in the Bst DNA polymerase is a conservative amino acidsubstitution. In further embodiments, the at least one conservativeamino acid substitution is selected from the group consisting ofhistidine to arginine, glutamic acid to glutamine, aspartic acid toasparagine, lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, the one or more conservative amino acidsubstitutions are of one or more amino acid residues shown in Table 2.In some embodiments, the one or more conservative amino acidsubstitutions are selected from the group consisting of H46R, H273R,H281R, E446Q, H473R, H528R, H572R and Y477F, the numbering of amino acidresidues being in accordance with that of SEQ ID NO: 1.

In some embodiments, the one or more amino acid substitutions includes asubstitution of alanine at position 2 with Met, Asn, Gln, Leu, Ile, Phe,or Trp, the numbering of amino acid residues being in accordance withthat of SEQ ID NO:2.

In some embodiments, the Bst DNA polymerase comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:3 and SEQ ID NO: 4, or a variant thereof having one or more conservativeamino acid substitutions. In some embodiments, the Bst DNA polymerasecomprises the amino acid sequence of SEQ ID NO: 2. In some embodiments,the Bst DNA polymerase is a variant of a protein comprising the aminoacid sequence shown in SEQ ID NO: 2, wherein the variant comprises anamino acid sequence that is at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 2.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 3. In some embodiments, the Bst DNA polymerase isa variant of a protein comprising the amino acid sequence shown in SEQID NO: 3, wherein the variant comprises an amino acid sequence that isat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 3.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 4. In other embodiments, the Bst polymerase is avariant of a protein comprising the amino acid sequence shown in SEQ IDNO: 4, wherein the variant comprises an amino acid sequence that is atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 4.

In some embodiments, the DNA polymerase is a Therminator™ DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In some embodiments, the DNA polymerase is a Therminator™ DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially remove its buffering capacity within the range of about pH7 to about pH 9, wherein the one or more conservative amino acidsubstitutions are selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In some embodiments, the DNA polymerase is a KOD DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table6.

In some embodiments, the DNA polymerase is a KOD DNA polymerasecomprising one or more amino acid substitutions that substantiallyremove its buffering capacity within the range of about pH 7 to about pH9. The one or more conservative amino acid substitutions are optionallyselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 6.

In some embodiments, the DNA polymerase is a B103 DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table7.

In some embodiments, the DNA polymerase is a B103 DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 4 toabout pH 10. The unsubstituted polymerase can be the wild-type versionof the polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table7.

In other embodiments of the method, the DNA polymerase is a B103 DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially remove its buffering capacity within the range ofabout pH 7 to about pH 9. The one or more conservative amino acidsubstitutions are optionally selected from the group consisting of:histidine to arginine, glutamic acid to glutamine, lysine to arginineand tyrosine to phenylalanine. In some embodiments, the one or moreconservative amino acid substitutions are of one or more amino acidresidues shown in Table 7.

In some embodiments, the disclosure relates generally to methods forsequencing a nucleic acid, comprising: (a) disposing a plurality oftemplate nucleic acids into a plurality of reaction chambers, whereinthe plurality of reaction chambers is in contact with achemical-sensitive field effect transistor (chemFET) array, the chemFETarray comprising at least one chemFET for each reaction chamber, andwherein each of the template nucleic acids is bound to a polymerase,wherein the polymerase comprises one or more amino acid substitutionsthat substantially reduce its buffering capacity; (b) synthesizing a newnucleic acid strand by sequentially incorporating one or more knownnucleoside triphosphates at the 3′ end of the sequencing primer, whereina hydrogen ion byproduct is generated when the known nucleosidetriphosphate is complementary to corresponding nucleotides in thetemplate nucleic acid; and (c) detecting the incorporation of the one ormore known nucleoside triphosphates by a change in voltage and/orcurrent at the at least one chemFET within the array in response to achange in hydrogen ion concentration proximate thereto.

In some embodiments, the one or more amino acid substitutions in thepolymerase may include any one or more of the amino acid substitutionsdescribed herein.

In some embodiments, at least one of the one or more amino acidsubstitutions can be a conservative amino acid substitution.

In some embodiments, each of the one or more amino acid substitutions isa conservative amino acid substitution.

In some embodiments, the polymerase includes any one of the modifiedpolymerases described herein. In some embodiments, the polymerase is abufferless polymerase. For example, the polymerase can have reducedbuffering capacity relative to the corresponding unsubstitutedpolymerase.

In some embodiments, the polymerase includes one or more amino acidsubstitutions that substantially remove the buffering capacity of thepolymerase within the pH range of about 4 to about 10 relative to thecorresponding unsubstituted polymerase. The unsubstituted polymerase canbe the wild-type version of the polymerase.

In some embodiments, the one or more amino acid substitutions in thepolymerase substantially remove the buffering capacity of the polymeraserelative to the corresponding unsubstituted polymerase within the rangeof about pH 7 to about pH 9. The unsubstituted polymerase can be thewild-type version of the polymerase.

In some embodiments, the polymerase includes one or more amino acidsubstitutions that substantially reduce the buffering capacity of thepolymerase within the pH range of about 4 to about 10 relative to thecorresponding unsubstituted polymerase. The unsubstituted polymerase canbe the wild-type version of the polymerase.

In some embodiments, the one or more amino acid substitutions in thepolymerase substantially reduce the buffering capacity of the polymeraserelative to the corresponding unsubstituted polymerase within the rangeof about pH 7 to about pH 9. The unsubstituted polymerase can be thewild-type version of the polymerase.

In some embodiments, at least one of the one or more amino acidsubstitutions in the polymerase is a conservative amino acidsubstitution that is selected from the group consisting of histidine toarginine, glutamic acid to glutamine, aspartic acid to asparagine,lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 4.0 to about 10.0 with another amino acidresidue. In some embodiments, the pKa of the amino acid residue is asolution pKa of the amino acid residue. In other embodiments, the pKa ofthe amino acid residue is a pKa of the amino acid residue in the contextof the corresponding wild-type protein.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 7 to about 9 with another amino acidresidue. In some embodiments, the pKa of the amino acid residue is asolution pKa of the amino acid residue. In other embodiments, the pKa ofthe amino acid residue is a pKa of the amino acid residue in the contextof the corresponding wild-type protein.

In some embodiments, at least one of the one or more conservative aminosubstitutions includes a substitution of an amino acid residue having apKa of between about 4.0 and about 10.0 with an amino acid residuehaving a pKa that is greater than about 10.0 or less than about 4.0. Infurther embodiments the amino acid residue having a pKa that is greaterthan about 10.0 or less than about 4.0 is selected from the groupconsisting of: Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe,Ser, Thr, Trp, Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more conservative aminosubstitutions includes a substitution of an amino acid residue having apKa of between about 7 and about 9 with an amino acid residue having apKa that is greater than about 9 or less than about 7. In furtherembodiments the amino acid residue having a pKa that is greater thanabout 9 or less than about 7 is selected from the group consisting of:Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp,Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 6.0 to about 8.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa of between about 6.0 and about 8.0 with an amino acid residue havinga pKa that is greater than about 8.0 or less than about 6.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 7.0 to about 9.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa of between about 7.0 and about 9.0 with an amino acid residue havinga pKa that is greater than about 9.0 or less than about 7.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue selectedfrom the group consisting of His, Glu, Asp, Tyr, and Lys with anotheramino acid residue.

In some embodiments, at least one of the one or more amino acidsubstitutions is a substitution of an amino acid residue with an alanineresidue.

In some embodiments, the polymerase comprises one or more conservativeamino acid substitutions that reduce the buffering capacity of theprotein relative to the corresponding wild-type protein within the rangeof about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, or about pH7 to about pH 9. In some embodiments, at least one of the one or moreamino acid substitutions includes a substitution of an amino acidresidue that is at least 20%, at least 25%, at least 30%, at least 35%or at least 40% solvent exposed in the corresponding wild-type proteinwith another amino acid residue.

In some embodiments, at least one of the one or more amino acidsubstitutions is a substitution of an amino acid residue with an alanineresidue.

In some embodiments, the polymerase comprises one or more amino acidconservative amino acid substitutions that reduce the buffering capacityof the protein relative to the corresponding wild-type protein withinthe range of about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, orabout pH 7 to about pH 9. In some embodiments, at least one of the oneor more amino acid substitutions includes a substitution of an aminoacid residue that is at least 20%, at least 25%, at least 30%, at least35% or at least 40% solvent exposed in the corresponding wild-typeprotein with another amino acid residue.

In some embodiments, the polymerase comprises one or more conservativeamino acid substitutions that substantially remove the bufferingcapacity of the polymerase within the range of about pH 4 to about pH10, about pH 5.5 to about pH 9.5, or about pH 7 to about pH 9.

In some embodiments, the polymerase is selected from the groupconsisting of an A family DNA polymerase; a B family DNA polymerase; amixed-type polymerase; an unclassified DNA polymerase and RT familypolymerase; and variants and derivatives thereof.

In some embodiments, the DNA polymerase is an A family DNA polymeraseselected from the group consisting of a Pol I-type DNA polymerase suchas E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, Bst DNA polymerase, Taq DNA polymerase, Platinum Taq DNApolymerase series, T7 DNA polymerase, and Tth DNA polymerase. In someembodiments, the DNA polymerase is Bst DNA polymerase. In otherembodiments, the DNA polymerase is E. coli DNA polymerase. In someembodiments, the DNA polymerase is the Klenow fragment of E. coli DNApolymerase. In some embodiments, the polymerase is Taq DNA polymerase.In some embodiments, the polymerase is T7 DNA polymerase.

In other embodiments, the DNA polymerase is a B family DNA polymeraseselected from the group consisting of Tli polymerase, Pfu polymerase,Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase,Sac polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, Pho polymerase, ES4 polymerase, VENT polymerase, DEEPVENTpolymerase, phage Phi29 polymerase, and phage B103 polymerase. In someembodiments, the polymerase is phage Phi29 DNA polymerase. In someembodiments the polymerase is phage B103 polymerase, including, forexample, the variants disclosed in U.S. Patent Publication No.20110014612.

In other embodiments, the DNA polymerase is a mixed-type polymeraseselected from the group consisting of EX-Taq polymerase, LA-Taqpolymerase, Expand polymerase series, and Hi-Fi polymerase. In yet otherembodiments, the DNA polymerase is an unclassified DNA polymeraseselected from the group consisting of Tbr polymerase, Tfl polymerase,Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tihpolymerase, and Tfi polymerase.

In other embodiments, the DNA polymerase is an RT polymerase selectedfrom the group consisting of HIV reverse transcriptase, M-MLV reversetranscriptase and AMV reverse transcriptase. In some embodiments, thepolymerase is HIV reverse transcriptase or a fragment thereof having DNApolymerase activity.

In some embodiments, the DNA polymerase is a Bst DNA polymerasecomprising one or more amino acid substitutions that substantiallyreduce its buffering capacity within the range of about pH 4 to about pH10. In some embodiments, the one or more amino acid substitutionssubstantially remove the buffering capacity of the polymerase within therange of about pH 4 to about pH 10.

In some embodiments, the one or more amino acid substitutions in the BstDNA polymerase substantially reduce the buffering capacity of thepolymerase relative to the corresponding unsubstituted polymerase withinthe range of about pH 7 to about pH 9. In some embodiments, the one ormore amino acid substitutions substantially remove the bufferingcapacity of the Bst polymerase within the range of about pH 7 to aboutpH 9. In some embodiments, the one or more amino acid substitutionssubstantially reduce the buffering capacity of the Bst polymeraserelative to the corresponding unsubstituted Bst polymerase within therange of about pH 7 to about pH 9. In some embodiments, theunsubstituted polymerase can be the wild-type version of the Bstpolymerase.

In some embodiments, at least one of the one or more amino acidsubstitutions in the Bst DNA polymerase is a conservative amino acidsubstitution. In further embodiments, the at least one conservativeamino acid substitution is selected from the group consisting ofhistidine to arginine, glutamic acid to glutamine, aspartic acid toasparagine, lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, the one or more conservative amino acidsubstitutions are of one or more amino acid residues shown in Table 2.In some embodiments, the one or more conservative amino acidsubstitutions are selected from the group consisting of H46R, H273R,H281R, E446Q, H473R, H528R, H572R and Y477F, the numbering of amino acidresidues being in accordance with that of SEQ ID NO: 1.

In some embodiments, the one or more amino acid substitutions includes asubstitution of alanine at position 2 with Met, Asn, Gln, Leu, Ile, Phe,or Trp, the numbering of amino acid residues being in accordance withthat of SEQ ID NO:2.

In some embodiments, the Bst DNA polymerase comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:3 and SEQ ID NO: 4, or a variant thereof having one or more conservativeamino acid substitutions. In some embodiments, the Bst DNA polymerasecomprises the amino acid sequence of SEQ ID NO: 2. In some embodiments,the Bst DNA polymerase is a variant of a protein comprising the aminoacid sequence shown in SEQ ID NO: 2, wherein the variant comprises anamino acid sequence that is at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 2.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 3. In some embodiments, the Bst DNA polymerase isa variant of a protein comprising the amino acid sequence shown in SEQID NO: 3, wherein the variant comprises an amino acid sequence that isat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 3.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 4. In other embodiments, the Bst polymerase is avariant of a protein comprising the amino acid sequence shown in SEQ IDNO: 4, wherein the variant comprises an amino acid sequence that is atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 4.

In some embodiments, the DNA polymerase is a Therminator™ DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In some embodiments, the DNA polymerase is a Therminator™ DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially remove its buffering capacity within the range of about pH7 to about pH 9, wherein the one or more conservative amino acidsubstitutions are selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In some embodiments, the DNA polymerase is a KOD DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table6.

In some embodiments, the DNA polymerase is a KOD DNA polymerasecomprising one or more amino acid substitutions that substantiallyremove its buffering capacity within the range of about pH 7 to about pH9. The one or more conservative amino acid substitutions are optionallyselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 6.

In some embodiments, the DNA polymerase is a B103 DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table7.

In some embodiments, the DNA polymerase is a B103 DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 4 toabout pH 10. The unsubstituted polymerase can be the wild-type versionof the polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table7.

In other embodiments of the method, the DNA polymerase is a B103 DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially remove its buffering capacity within the range ofabout pH 7 to about pH 9. The one or more conservative amino acidsubstitutions are optionally selected from the group consisting of:histidine to arginine, glutamic acid to glutamine, lysine to arginineand tyrosine to phenylalanine. In some embodiments, the one or moreconservative amino acid substitutions are of one or more amino acidresidues shown in Table 7.

In some embodiments, the disclosure relates generally to methods forsequencing a nucleic acid, comprising: (a) disposing a plurality ofsolid supports into a plurality of reaction chambers on a sensor arrayformed in a semiconductor substrate, each reaction chamber comprising asingle solid support, each solid support attached to a plurality ofidentical template nucleic acids, each of the template nucleic acidshybridized to a sequencing primer and bound to a polymerase, wherein thepolymerase comprises one or more amino acid substitutions thatsubstantially reduce its buffering capacity, and each reaction chamberin contact with or capacitavely coupled to at least one chemFET of asensor, and each such chemFET being configured to provide at least oneoutput representing the presence and/or concentration of hydrogen ionproximate thereto; (b) introducing a known nucleoside triphosphate intoeach reaction chamber; (c) detecting sequential incorporation at the 3′end of the sequencing primer of one or more nucleoside triphosphates bythe generation of a change in hydrogen ion concentration when the knownnucleoside triphosphate is complementary to corresponding nucleotides inthe template nucleic acid; and (d) washing unincorporated nucleosidetriphosphates from the reaction chambers.

In some embodiments, the method further includes (e) repeating steps (b)through (d) until the nucleic acid is sequenced.

In some embodiments, the one or more amino acid substitutions in thepolymerase may include any one or more of the amino acid substitutionsdescribed herein.

In some embodiments, at least one of the one or more amino acidsubstitutions can be a conservative amino acid substitution.

In some embodiments, each of the one or more amino acid substitutions isa conservative amino acid substitution.

In some embodiments, the polymerase includes any one of the modifiedpolymerases described herein. In some embodiments, the polymerase is abufferless polymerase. For example, the polymerase can have reducedbuffering capacity relative to the corresponding unsubstitutedpolymerase.

In some embodiments, the polymerase includes one or more amino acidsubstitutions that substantially remove the buffering capacity of thepolymerase within the pH range of about 4 to about 10 relative to thecorresponding unsubstituted polymerase. The unsubstituted polymerase canbe the wild-type version of the polymerase.

In some embodiments, the one or more amino acid substitutions in thepolymerase substantially remove the buffering capacity of the polymeraserelative to the corresponding unsubstituted polymerase within the rangeof about pH 7 to about pH 9. The unsubstituted polymerase can be thewild-type version of the polymerase.

In some embodiments, the polymerase includes one or more amino acidsubstitutions that substantially reduce the buffering capacity of thepolymerase within the pH range of about 4 to about 10 relative to thecorresponding unsubstituted polymerase. The unsubstituted polymerase canbe the wild-type version of the polymerase.

In some embodiments, the one or more amino acid substitutions in thepolymerase substantially reduce the buffering capacity of the polymeraserelative to the corresponding unsubstituted polymerase within the rangeof about pH 7 to about pH 9. The unsubstituted polymerase can be thewild-type version of the polymerase.

In some embodiments, at least one of the one or more amino acidsubstitutions in the polymerase is a conservative amino acidsubstitution that is selected from the group consisting of histidine toarginine, glutamic acid to glutamine, aspartic acid to asparagine,lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 4.0 to about 10.0 with another amino acidresidue. In some embodiments, the pKa of the amino acid residue is asolution pKa of the amino acid residue. In other embodiments, the pKa ofthe amino acid residue is a pKa of the amino acid residue in the contextof the corresponding wild-type protein.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 7 to about 9 with another amino acidresidue. In some embodiments, the pKa of the amino acid residue is asolution pKa of the amino acid residue. In other embodiments, the pKa ofthe amino acid residue is a pKa of the amino acid residue in the contextof the corresponding wild-type protein.

In some embodiments, at least one of the one or more conservative aminosubstitutions includes a substitution of an amino acid residue having apKa of between about 4.0 and about 10.0 with an amino acid residuehaving a pKa that is greater than about 10.0 or less than about 4.0. Infurther embodiments the amino acid residue having a pKa that is greaterthan about 10.0 or less than about 4.0 is selected from the groupconsisting of: Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe,Ser, Thr, Trp, Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more conservative aminosubstitutions includes a substitution of an amino acid residue having apKa of between about 7 and about 9 with an amino acid residue having apKa that is greater than about 9 or less than about 7. In furtherembodiments the amino acid residue having a pKa that is greater thanabout 9 or less than about 7 is selected from the group consisting of:Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp,Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 6.0 to about 8.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa of between about 6.0 and about 8.0 with an amino acid residue havinga pKa that is greater than about 8.0 or less than about 6.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 7.0 to about 9.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa of between about 7.0 and about 9.0 with an amino acid residue havinga pKa that is greater than about 9.0 or less than about 7.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue selectedfrom the group consisting of His, Glu, Asp, Tyr, and Lys with anotheramino acid residue.

In some embodiments, at least one of the one or more amino acidsubstitutions is a substitution of an amino acid residue with an alanineresidue.

In some embodiments, the polymerase comprises one or more conservativeamino acid substitutions that reduce the buffering capacity of theprotein relative to the corresponding wild-type protein within the rangeof about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, or about pH7 to about pH 9. In some embodiments, at least one of the one or moreamino acid substitutions includes a substitution of an amino acidresidue that is at least 20%, at least 25%, at least 30%, at least 35%or at least 40% solvent exposed in the corresponding wild-type proteinwith another amino acid residue.

In some embodiments, at least one of the one or more amino acidsubstitutions is a substitution of an amino acid residue with an alanineresidue.

In some embodiments, the polymerase comprises one or more amino acidconservative amino acid substitutions that reduce the buffering capacityof the protein relative to the corresponding wild-type protein withinthe range of about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, orabout pH 7 to about pH 9. In some embodiments, at least one of the oneor more amino acid substitutions includes a substitution of an aminoacid residue that is at least 20%, at least 25%, at least 30%, at least35% or at least 40% solvent exposed in the corresponding wild-typeprotein with another amino acid residue.

In some embodiments, the polymerase comprises one or more conservativeamino acid substitutions that substantially remove the bufferingcapacity of the polymerase within the range of about pH 4 to about pH10, about pH 5.5 to about pH 9.5, or about pH 7 to about pH 9.

In some embodiments, the polymerase is selected from the groupconsisting of an A family DNA polymerase; a B family DNA polymerase; amixed-type polymerase; an unclassified DNA polymerase and RT familypolymerase; and variants and derivatives thereof.

In some embodiments, the DNA polymerase is an A family DNA polymeraseselected from the group consisting of a Pol I-type DNA polymerase suchas E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, Bst DNA polymerase, Taq DNA polymerase, Platinum Taq DNApolymerase series, T7 DNA polymerase, and Tth DNA polymerase. In someembodiments, the DNA polymerase is Bst DNA polymerase. In otherembodiments, the DNA polymerase is E. coli DNA polymerase. In someembodiments, the DNA polymerase is the Klenow fragment of E. coli DNApolymerase. In some embodiments, the polymerase is Taq DNA polymerase.In some embodiments, the polymerase is T7 DNA polymerase.

In other embodiments, the DNA polymerase is a B family DNA polymeraseselected from the group consisting of Tli polymerase, Pfu polymerase,Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase,Sac polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, Pho polymerase, ES4 polymerase, VENT polymerase, DEEPVENTpolymerase, phage Phi29 polymerase, and phage B103 polymerase. In someembodiments, the polymerase is phage Phi29 DNA polymerase. In someembodiments the polymerase is phage B103 polymerase, including, forexample, the variants disclosed in U.S. Patent Publication No.20110014612.

In other embodiments, the DNA polymerase is a mixed-type polymeraseselected from the group consisting of EX-Taq polymerase, LA-Taqpolymerase, Expand polymerase series, and Hi-Fi polymerase. In yet otherembodiments, the DNA polymerase is an unclassified DNA polymeraseselected from the group consisting of Tbr polymerase, Tfl polymerase,Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tihpolymerase, and Tfi polymerase.

In other embodiments, the DNA polymerase is an RT polymerase selectedfrom the group consisting of HIV reverse transcriptase, M-MLV reversetranscriptase and AMV reverse transcriptase. In some embodiments, thepolymerase is HIV reverse transcriptase or a fragment thereof having DNApolymerase activity.

In some embodiments, the DNA polymerase is a Bst DNA polymerasecomprising one or more amino acid substitutions that substantiallyreduce its buffering capacity within the range of about pH 4 to about pH10. In some embodiments, the one or more amino acid substitutionssubstantially remove the buffering capacity of the polymerase within therange of about pH 4 to about pH 10.

In some embodiments, the one or more amino acid substitutions in the BstDNA polymerase substantially reduce the buffering capacity of thepolymerase relative to the corresponding unsubstituted polymerase withinthe range of about pH 7 to about pH 9. In some embodiments, the one ormore amino acid substitutions substantially remove the bufferingcapacity of the Bst polymerase within the range of about pH 7 to aboutpH 9. In some embodiments, the one or more amino acid substitutionssubstantially reduce the buffering capacity of the Bst polymeraserelative to the corresponding unsubstituted Bst polymerase within therange of about pH 7 to about pH 9. In some embodiments, theunsubstituted polymerase can be the wild-type version of the Bstpolymerase.

In some embodiments, at least one of the one or more amino acidsubstitutions in the Bst DNA polymerase is a conservative amino acidsubstitution. In further embodiments, the at least one conservativeamino acid substitution is selected from the group consisting ofhistidine to arginine, glutamic acid to glutamine, aspartic acid toasparagine, lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, the one or more conservative amino acidsubstitutions are of one or more amino acid residues shown in Table 2.In some embodiments, the one or more conservative amino acidsubstitutions are selected from the group consisting of H46R, H273R,H281R, E446Q, H473R, H528R, H572R and Y477F, the numbering of amino acidresidues being in accordance with that of SEQ ID NO: 1.

In some embodiments, the one or more amino acid substitutions includes asubstitution of alanine at position 2 with Met, Asn, Gln, Leu, Ile, Phe,or Trp, the numbering of amino acid residues being in accordance withthat of SEQ ID NO:2.

In some embodiments, the Bst DNA polymerase comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:3 and SEQ ID NO: 4, or a variant thereof having one or more conservativeamino acid substitutions. In some embodiments, the Bst DNA polymerasecomprises the amino acid sequence of SEQ ID NO: 2. In some embodiments,the Bst DNA polymerase is a variant of a protein comprising the aminoacid sequence shown in SEQ ID NO: 2, wherein the variant comprises anamino acid sequence that is at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 2.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 3. In some embodiments, the Bst DNA polymerase isa variant of a protein comprising the amino acid sequence shown in SEQID NO: 3, wherein the variant comprises an amino acid sequence that isat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 3.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 4. In other embodiments, the Bst polymerase is avariant of a protein comprising the amino acid sequence shown in SEQ IDNO: 4, wherein the variant comprises an amino acid sequence that is atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 4.

In some embodiments, the DNA polymerase is a Therminator™ DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In some embodiments, the DNA polymerase is a Therminator™ DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially remove its buffering capacity within the range of about pH7 to about pH 9, wherein the one or more conservative amino acidsubstitutions are selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In some embodiments, the DNA polymerase is a KOD DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table6.

In some embodiments, the DNA polymerase is a KOD DNA polymerasecomprising one or more amino acid substitutions that substantiallyremove its buffering capacity within the range of about pH 7 to about pH9. The one or more conservative amino acid substitutions are optionallyselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 6.

In some embodiments, the DNA polymerase is a B103 DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table7.

In some embodiments, the DNA polymerase is a B103 DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 4 toabout pH 10. The unsubstituted polymerase can be the wild-type versionof the polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table7.

In other embodiments of the method, the DNA polymerase is a B103 DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially remove its buffering capacity within the range ofabout pH 7 to about pH 9. The one or more conservative amino acidsubstitutions are optionally selected from the group consisting of:histidine to arginine, glutamic acid to glutamine, lysine to arginineand tyrosine to phenylalanine. In some embodiments, the one or moreconservative amino acid substitutions are of one or more amino acidresidues shown in Table 7.

In some embodiments, the disclosure relates generally to methods forsequencing a nucleic acid, comprising: (a) disposing a solid supportinto a reaction chamber on a sensor array formed in a semiconductorsubstrate, where the solid support is attached to a plurality ofsubstantially identical template nucleic acids, at least one of thetemplate nucleic acids being hybridized to a sequencing primer and boundto a polymerase, wherein the polymerase comprises one or more amino acidsubstitutions that substantially reduce its buffering capacity, and thereaction chamber is in contact with or capacitavely coupled to at leastone chemFET of a sensor, the chemFET being configured to provide atleast one output representing the presence and/or concentration ofhydrogen ion proximate thereto; (b) introducing nucleoside triphosphatesof a known type into the reaction chamber; (c) detecting incorporationat the 3′end of the sequencing primer of one or more nucleosidetriphosphates of the known type by detecting the generation of a changein hydrogen ion concentration resulting from the incorporation of theone or more nucleoside triphosphates by the polymerase; and (d) washingunincorporated nucleoside triphosphates from the reaction chamber.

In some embodiments, the method further includes (e) repeating steps (b)through (d) until the nucleic acid is sequenced.

In some embodiments, the one or more amino acid substitutions in thepolymerase may include any one or more of the amino acid substitutionsdescribed herein.

In some embodiments, at least one of the one or more amino acidsubstitutions can be a conservative amino acid substitution.

In some embodiments, each of the one or more amino acid substitutions isa conservative amino acid substitution.

In some embodiments, the polymerase includes any one of the modifiedpolymerases described herein. In some embodiments, the polymerase is abufferless polymerase. For example, the polymerase can have reducedbuffering capacity relative to the corresponding unsubstitutedpolymerase.

In some embodiments, the polymerase includes one or more amino acidsubstitutions that substantially remove the buffering capacity of thepolymerase within the pH range of about 4 to about 10 relative to thecorresponding unsubstituted polymerase. The unsubstituted polymerase canbe the wild-type version of the polymerase.

In some embodiments, the one or more amino acid substitutions in thepolymerase substantially remove the buffering capacity of the polymeraserelative to the corresponding unsubstituted polymerase within the rangeof about pH 7 to about pH 9. The unsubstituted polymerase can be thewild-type version of the polymerase.

In some embodiments, the polymerase includes one or more amino acidsubstitutions that substantially reduce the buffering capacity of thepolymerase within the pH range of about 4 to about 10 relative to thecorresponding unsubstituted polymerase. The unsubstituted polymerase canbe the wild-type version of the polymerase.

In some embodiments, the one or more amino acid substitutions in thepolymerase substantially reduce the buffering capacity of the polymeraserelative to the corresponding unsubstituted polymerase within the rangeof about pH 7 to about pH 9. The unsubstituted polymerase can be thewild-type version of the polymerase.

In some embodiments, at least one of the one or more amino acidsubstitutions in the polymerase is a conservative amino acidsubstitution that is selected from the group consisting of histidine toarginine, glutamic acid to glutamine, aspartic acid to asparagine,lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 4.0 to about 10.0 with another amino acidresidue. In some embodiments, the pKa of the amino acid residue is asolution pKa of the amino acid residue. In other embodiments, the pKa ofthe amino acid residue is a pKa of the amino acid residue in the contextof the corresponding wild-type protein.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 7 to about 9 with another amino acidresidue. In some embodiments, the pKa of the amino acid residue is asolution pKa of the amino acid residue. In other embodiments, the pKa ofthe amino acid residue is a pKa of the amino acid residue in the contextof the corresponding wild-type protein.

In some embodiments, at least one of the one or more conservative aminosubstitutions includes a substitution of an amino acid residue having apKa of between about 4.0 and about 10.0 with an amino acid residuehaving a pKa that is greater than about 10.0 or less than about 4.0. Infurther embodiments the amino acid residue having a pKa that is greaterthan about 10.0 or less than about 4.0 is selected from the groupconsisting of: Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe,Ser, Thr, Trp, Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more conservative aminosubstitutions includes a substitution of an amino acid residue having apKa of between about 7 and about 9 with an amino acid residue having apKa that is greater than about 9 or less than about 7. In furtherembodiments the amino acid residue having a pKa that is greater thanabout 9 or less than about 7 is selected from the group consisting of:Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp,Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 6.0 to about 8.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa of between about 6.0 and about 8.0 with an amino acid residue havinga pKa that is greater than about 8.0 or less than about 6.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 7.0 to about 9.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa of between about 7.0 and about 9.0 with an amino acid residue havinga pKa that is greater than about 9.0 or less than about 7.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue selectedfrom the group consisting of His, Glu, Asp, Tyr, and Lys with anotheramino acid residue.

In some embodiments, at least one of the one or more amino acidsubstitutions is a substitution of an amino acid residue with an alanineresidue.

In some embodiments, the polymerase comprises one or more conservativeamino acid substitutions that reduce the buffering capacity of theprotein relative to the corresponding wild-type protein within the rangeof about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, or about pH7 to about pH 9. In some embodiments, at least one of the one or moreamino acid substitutions includes a substitution of an amino acidresidue that is at least 20%, at least 25%, at least 30%, at least 35%or at least 40% solvent exposed in the corresponding wild-type proteinwith another amino acid residue.

In some embodiments, at least one of the one or more amino acidsubstitutions is a substitution of an amino acid residue with an alanineresidue.

In some embodiments, the polymerase comprises one or more amino acidconservative amino acid substitutions that reduce the buffering capacityof the protein relative to the corresponding wild-type protein withinthe range of about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, orabout pH 7 to about pH 9. In some embodiments, at least one of the oneor more amino acid substitutions includes a substitution of an aminoacid residue that is at least 20%, at least 25%, at least 30%, at least35% or at least 40% solvent exposed in the corresponding wild-typeprotein with another amino acid residue.

In some embodiments, the polymerase comprises one or more conservativeamino acid substitutions that substantially remove the bufferingcapacity of the polymerase within the range of about pH 4 to about pH10, about pH 5.5 to about pH 9.5, or about pH 7 to about pH 9.

In some embodiments, the polymerase is selected from the groupconsisting of an A family DNA polymerase; a B family DNA polymerase; amixed-type polymerase; an unclassified DNA polymerase and RT familypolymerase; and variants and derivatives thereof.

In some embodiments, the DNA polymerase is an A family DNA polymeraseselected from the group consisting of a Pol I-type DNA polymerase suchas E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, Bst DNA polymerase, Taq DNA polymerase, Platinum Taq DNApolymerase series, T7 DNA polymerase, and Tth DNA polymerase. In someembodiments, the DNA polymerase is Bst DNA polymerase. In otherembodiments, the DNA polymerase is E. coli DNA polymerase. In someembodiments, the DNA polymerase is the Klenow fragment of E. coli DNApolymerase. In some embodiments, the polymerase is Taq DNA polymerase.In some embodiments, the polymerase is T7 DNA polymerase.

In other embodiments, the DNA polymerase is a B family DNA polymeraseselected from the group consisting of Tli polymerase, Pfu polymerase,Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase,Sac polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, Pho polymerase, ES4 polymerase, VENT polymerase, DEEPVENTpolymerase, phage Phi29 polymerase, and phage B103 polymerase. In someembodiments, the polymerase is phage Phi29 DNA polymerase. In someembodiments the polymerase is phage B103 polymerase, including, forexample, the variants disclosed in U.S. Patent Publication No.20110014612.

In other embodiments, the DNA polymerase is a mixed-type polymeraseselected from the group consisting of EX-Taq polymerase, LA-Taqpolymerase, Expand polymerase series, and Hi-Fi polymerase. In yet otherembodiments, the DNA polymerase is an unclassified DNA polymeraseselected from the group consisting of Tbr polymerase, Tfl polymerase,Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tihpolymerase, and Tfi polymerase.

In other embodiments, the DNA polymerase is an RT polymerase selectedfrom the group consisting of HIV reverse transcriptase, M-MLV reversetranscriptase and AMV reverse transcriptase. In some embodiments, thepolymerase is HIV reverse transcriptase or a fragment thereof having DNApolymerase activity.

In some embodiments, the DNA polymerase is a Bst DNA polymerasecomprising one or more amino acid substitutions that substantiallyreduce its buffering capacity within the range of about pH 4 to about pH10. In some embodiments, the one or more amino acid substitutionssubstantially remove the buffering capacity of the polymerase within therange of about pH 4 to about pH 10.

In some embodiments, the one or more amino acid substitutions in the BstDNA polymerase substantially reduce the buffering capacity of thepolymerase relative to the corresponding unsubstituted polymerase withinthe range of about pH 7 to about pH 9. In some embodiments, the one ormore amino acid substitutions substantially remove the bufferingcapacity of the Bst polymerase within the range of about pH 7 to aboutpH 9. In some embodiments, the one or more amino acid substitutionssubstantially reduce the buffering capacity of the Bst polymeraserelative to the corresponding unsubstituted Bst polymerase within therange of about pH 7 to about pH 9. In some embodiments, theunsubstituted polymerase can be the wild-type version of the Bstpolymerase.

In some embodiments, at least one of the one or more amino acidsubstitutions in the Bst DNA polymerase is a conservative amino acidsubstitution. In further embodiments, the at least one conservativeamino acid substitution is selected from the group consisting ofhistidine to arginine, glutamic acid to glutamine, aspartic acid toasparagine, lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, the one or more conservative amino acidsubstitutions are of one or more amino acid residues shown in Table 2.In some embodiments, the one or more conservative amino acidsubstitutions are selected from the group consisting of H46R, H273R,H281R, E446Q, H473R, H528R, H572R and Y477F, the numbering of amino acidresidues being in accordance with that of SEQ ID NO: 1.

In some embodiments, the one or more amino acid substitutions includes asubstitution of alanine at position 2 with Met, Asn, Gln, Leu, Ile, Phe,or Trp, the numbering of amino acid residues being in accordance withthat of SEQ ID NO:2.

In some embodiments, the Bst DNA polymerase comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:3 and SEQ ID NO: 4, or a variant thereof having one or more conservativeamino acid substitutions. In some embodiments, the Bst DNA polymerasecomprises the amino acid sequence of SEQ ID NO: 2. In some embodiments,the Bst DNA polymerase is a variant of a protein comprising the aminoacid sequence shown in SEQ ID NO: 2, wherein the variant comprises anamino acid sequence that is at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 2.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 3. In some embodiments, the Bst DNA polymerase isa variant of a protein comprising the amino acid sequence shown in SEQID NO: 3, wherein the variant comprises an amino acid sequence that isat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 3.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 4. In other embodiments, the Bst polymerase is avariant of a protein comprising the amino acid sequence shown in SEQ IDNO: 4, wherein the variant comprises an amino acid sequence that is atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 4.

In some embodiments, the DNA polymerase is a Therminator™ DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In some embodiments, the DNA polymerase is a Therminator™ DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially remove its buffering capacity within the range of about pH7 to about pH 9, wherein the one or more conservative amino acidsubstitutions are selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In some embodiments, the DNA polymerase is a KOD DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table6.

In some embodiments, the DNA polymerase is a KOD DNA polymerasecomprising one or more amino acid substitutions that substantiallyremove its buffering capacity within the range of about pH 7 to about pH9. The one or more conservative amino acid substitutions are optionallyselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 6.

In some embodiments, the DNA polymerase is a B103 DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table7.

In some embodiments, the DNA polymerase is a B103 DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 4 toabout pH 10. The unsubstituted polymerase can be the wild-type versionof the polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table7.

In other embodiments of the method, the DNA polymerase is a B103 DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially remove its buffering capacity within the range ofabout pH 7 to about pH 9. The one or more conservative amino acidsubstitutions are optionally selected from the group consisting of:histidine to arginine, glutamic acid to glutamine, lysine to arginineand tyrosine to phenylalanine. In some embodiments, the one or moreconservative amino acid substitutions are of one or more amino acidresidues shown in Table 7.

Any suitable method of performing nucleic acid synthesis involvingnucleotide incorporation and/or primer extension using a polymerase maybe used to practice, use or perform any of the disclosed methods,compositions, systems, apparatuses and kits. Typically, polymerases arecapable of catalyzing nucleotide incorporation onto the terminal 3′OHend of an extending nucleic acid molecule. When the extending nucleicacid molecule comprises a primer, the process is typically referred toas “primer extension.” Typically but not necessarily such nucleotideincorporation occurs in a template-dependent fashion. Primer extensionand other nucleotide incorporation assays are typically performed bycontacting the template nucleic acid with a polymerase in the presenceof nucleotides in an aqueous solution under nucleotide incorporationconditions. In some embodiments, the nucleotide incorporation reactioncan include a primer, which can optionally be hybridized to the templateto form a primer-template duplex. Typical nucleotide incorporationconditions are achieved once the template, polymerase, nucleotides andoptionally primer are mixed with each other in a suitable aqueousformulation, thereby forming a nucleotide incorporation reaction mixture(or primer extension mixture). The aqueous formation can optionallyinclude divalent cations and/or salts, particularly Mg⁺⁺ and/or Ca⁺⁺ions. Typical nucleotide incorporation conditions have included wellknown parameters for time, temperature, pH, reagents, buffers, reagents,salts, co-factors, nucleotides, target DNA, primer DNA, enzymes such asnucleic acid-dependent polymerase, amounts and/or ratios of thecomponents in the reactions, and the like. The reagents or buffers caninclude a source of monovalent ions, such as KCl, K-acetate,NH₄-acetate, K-glutamate, NH₄Cl, or ammonium sulfate. The reagents orbuffers can include a source of divalent ions, such as Mg²⁺ and/or Mn²⁺,MgCl₂, or Mg-acetate. Most polymerases exhibit some levels of nucleotideincorporation activity over pH range of about 5.0 to about 9.5, moretypically between about pH 7 and about pH 9, and sometimes between aboutpH 6 to about pH 8. The buffer can include chelating agents such as EDTAand EGTA, and the like. Although in some embodiments, nucleotideincorporation reactions may include buffering agents, such as Tris,Tricine, HEPES, MOPS, ACES, or MES, which can provide a pH range ofabout 5.0 to about 9.5, such buffering agents can optionally be reducedor eliminated when performing ion-based reactions requiring detection ofion byproducts. Methods of performing nucleic acid synthesis are wellknown and extensively practiced in the art and references teaching awide range of nucleic acid synthesis techniques are readily available.Some exemplary teachings regarding the performance of nucleic acidsynthesis (including, for example, template-dependent nucleotideincorporation, as well as primer extension methods) can be found, forexample, in Kim et al., Nature 376: 612-616 (2002); Ichida et al.,Nucleic Acids Res. 33: 5214-5222 (2005); Pandey et al., European Journalof Biochemistry, 214:59-65 (1993); Blanco et al., J. Biol. Chem. 268:16763-16770 (1993); U.S. patent application Ser. No. 12/002,781, nowpublished as U.S. Patent Publication No. 2009/0026082; U.S. patentapplication Ser. No. 12/474,897, now published as U.S. PatentPublication No. 2010/0137143; and U.S. patent application Ser. No.12/492,844, now published as U.S. Patent Publication No. 2010/0282617;U.S. patent application Ser. No. 12/748,359, now published as U.S.Patent Publication No. 20110014612.

In some embodiments, the disclosure also relates to modes for analyzing,including for example sequencing, nucleic acids using reactions thatinvolve interdependent nucleotide incorporation and nucleotide excision.As used herein, interdependent nucleotide incorporation and nucleotideexcision means that both reactions occur on the same nucleic molecule atcontiguous sites on the nucleic acid, and one reaction facilitates theother. An example of such a reaction is a nick translation reaction. Anick translation reaction, as used herein, refers to a reactioncatalyzed by a polymerase enzyme having 5′ to 3′ exonuclease activity,that involves incorporation of a nucleotide onto the free 3′ end of anicked region of double stranded DNA and excision of a nucleotidelocated at the free 5′ end of the nicked region of the double strandedDNA. Nick translation therefore refers to the movement of the nickedsite along the length of the nicked strand of DNA in a 5′ to 3′direction. As will be recognized by those of ordinary skill in the art,the nick translation reaction includes a sequencing-by-synthesisreaction based on the intact strand of the double stranded DNA. Thisstrand acts as the template from which the new strand is synthesized.The method does not require the use of a primer because the doublestranded DNA can prime the reaction independently. While the disclosure(including the following passages) may often refer to “nick translation”for the sake of brevity, it is to be understood that the scope of thisdisclosure includes any other combined reaction of nucleotide excisionand incorporation to be used in place of traditional nick translation.

The nick translation approach has two features that make it well suitedfor use in the nucleotide incorporation and sequencing methods providedherein. First, the nick translation reaction results in the release oftwo hydrogen ions for each combined excision/incorporation step, therebyproviding a more robust signal at the chemFET each time a nucleotide isincorporated into a newly synthesized strand. A sequencing-by-synthesismethod, in the absence of nucleotide excision, releases one hydrogen ionper nucleotide incorporation. In contrast, nick translation releases afirst hydrogen ion upon incorporation of a nucleotide and a secondhydrogen ion upon excision of another nucleotide. This increases thesignal that can be sensed at the chemFET, thereby increasing signal tonoise ratio and providing a more definitive readout of nucleotideincorporation.

Second, the use of a double stranded DNA template (rather than a singlestranded DNA template) results in less interference of the template withreleased ions and a better signal at the chemFET. A single stranded DNAhas exposed groups that are able to interfere with (for example,sequester) hydrogen ions. These reactive groups are shielded in a doublestranded DNA where they are hydrogen bonded to complementary groups. Bybeing so shielded, these groups do not substantially impact hydrogen ionlevel or concentration. As a result, signal resulting from hydrogen ionrelease is greater in the presence of double stranded as compared tosingle stranded templates, as will be signal to noise ratio, therebyfurther contributing to a more definitive readout of nucleotideincorporation.

Templates suitable for nick translation typically are completely orpartially double stranded. Such templates comprise an opening (or anick) which acts as an entry point for a polymerase. Such openings canbe introduced into the template in a controlled manner as describedbelow and known in the art.

As will be appreciated by one of ordinary skill in the art, it ispreferable that these openings be present in each of the plurality ofidentical templates at the same location in the template sequence.Typical molecular biology techniques involving nick translation userandomly created nicks along the double stranded DNA because their aimis to produce a detectably labeled nucleic acid. These prior art methodsgenerate nicks through the use of sequence-independent nicking enzymessuch as DNase I. In many methods, however, the nick location must beknown, non-random and uniform for all templates of identical sequence.Various ways of achieving this are known in the art, and some of theseare discussed below by way of non-limiting examples.

Another example of a suitable nick translation template is aself-priming nucleic acid. The self priming nucleic acid may comprise adouble stranded and a single stranded region that is capable ofself-annealing in order to prime a nucleic acid synthesis reaction. Thesingle stranded region is typically a known synthetic sequence ligatedto a nucleic acid of interest. Its length can be predetermined andengineered to create an opening following self-annealing, and suchopening can act as an entry point for a polymerase.

It is to be understood that, as the term is used herein, a nickednucleic acid, such as a nicked double stranded nucleic acid, is anucleic acid having an opening (e.g., a break in its backbone, or havingabasic sites, etc.) from which a polymerase can incorporate andoptionally excise nucleotides. The term is not limited to nucleic acidsthat have been acted upon by an enzyme such as a nicking enzyme, nor isit limited simply to breaks in a nucleic acid backbone, as will be clearbased on the exemplary methods described herein for creating suchnucleic acids.

Once the nicked double stranded nucleic acids are generated, they arethen subjected to a nick translation reaction. If the nick translationreaction is performed to sequence the template nucleic acid, the nicktranslation can be carried out in a manner that parallels thesequencing-by-synthesis methods described herein. More specifically, insome embodiments each of the four nucleotides is separately contactedwith the nicked templates in the presence of a polymerase having 5′ to3′ exonuclease activity. In other embodiments, known combinations ofnucleotides are used. Examples of suitable enzymes include DNApolymerase I from E. coli, Bst DNA polymerase, and Taq DNA polymerase.The order of the nucleotides is not important as long as it is known andpreferably remains the same throughout a run. After each nucleotide iscontacted with the nicked templates, it is washed out followed by theintroduction of another nucleotide, just as described herein. In thenick translation embodiments, the wash will also carry the excisednucleotide away from the chemFET.

It should be appreciated that just as with other aspects and embodimentsdescribed herein the nucleotides that are incorporated into the nickedregion need not be extrinsically labeled since it is a byproduct oftheir incorporation that is detected as a readout rather than theincorporated nucleotide itself. Thus, the nick translation methods maybe referred to as label-free methods, or fluorescence-free methods,since incorporation detection is not dependent on an extrinsic label onthe incorporated nucleotide. The nucleotides are typically naturallyoccurring nucleotides. It should also be recognized that since themethods benefit from the consecutive incorporation of as manynucleotides as possible, the nucleotides are not for example modifiedversions that lead to premature chain termination, such as those used insome sequencing methods.

EXAMPLES Example 1. Design and Expression of Modified Bst DNAPolymerases

The amino acid sequence of the large fragment of the Bst DNA polymerase,having the amino acid sequencing of SEQ ID NO: 1, was analyzed usingboth H++ and PropKa to calculate the pKas of titratable side chainswithin the folded protein structure. Tables 2 and 3 show the calculatedpKas for amino acids within Bst DNA polymerase. Column 1 shows the aminoacid residue, with numbering based upon that of SEQ ID NO:1; column 2shows the calculated pKa of the side chain. Column 3 indicates whetherthe residue is accessible on the surface of the protein (S) or buried(B), as determined by a review of the protein structure. The valuesshown in Table 2 were determined using PropKa, while the values in Table3 were determined using H++.

Amino acid residues having calculated pKas within the range of about pH6 to about pH 8.0 as determined by either algorithm were targeted forsubstitution. A nucleic acid sequence encoding the large fragment of BstDNA polymerase (having the amino acid sequence of SEQ ID NO: 1) wasmutated using conventional techniques to introduce the following aminoacid substitutions into the encoded protein product: His46Arg,Glu446Gln, and His572Arg. The resulting amino acid sequence is shown inSEQ ID NO: 2. The substituted amino acids are underlined and in bold.

SEQ ID NO: 2 MAKMAFTLADRVTEEMLADKAALVVEVVEENYHDAPIVGIAVVNE R GRFFLRPETALADPQFVAWLGDETKKKSMFDSKRAAVALKWKGIELCGVSFDLLLAAYLLDPAQGVDDVAAAAKMKQYEAVRPDEAVYGKGAKRAVPDEPVLAEHLVRKAAAIWELERPFLDELRRNEQDRLLVELEQPLSSILAEMEFAGVKVDTKRLEQMGKELAEQLGTVEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPYHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTKKVHTIFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSESDWLIFAADYSQIELRVLAHIAEDDNLMEAFRRDLDIHTKTAMDIFQVSEDEVTPNMRRQAKAVNFGIVYGISDYGLAQNLNISRKEAAEFIERYF Q SFPGVKRYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERMAMNTPIQGSAADIIKKAMIDLNARLKEERLQAHLLLQVHDELILEAPKEEMERLC RLVPEVMEQAVTLRVPLKVDYR YGSTWYDAK

The nucleic acid sequence encoding SEQ ID NO: 2 was further mutatedusing conventional techniques to create nucleic acid sequences encodingpolymerases having additional mutations. SEQ ID NO: 3 contains twofurther amino acid substitutions as compared to SEQ ID NO:2: His473Argand His528Ala. Thus SEQ ID NO: 3 contains a total of five amino acidsubstitutions as compared to the reference wild-type sequence of SEQ IDNO:1. The substituted amino acids as compared to SEQ ID NO: 1 areunderlined and in bold.

SEQ ID NO: 3 MAKMAFTLADRVITEMLADKAALVVEVVEENYHDAPIVGIAVVNE R GRFFLRPETALADPQFVAWLGDETKKKSMFDSKRAAVALKWKGIELCGVSFDLLLAAYLLDPAQGVDDVAAAAKMKQYEAVRPDEAVYGKGAKRAVPDEPVLAEHLVRKAAAIWELERPFLDELRRNEQDRLLVELEQPLSSILAEMEFAGVKVDTKRLEQMGKELAEQLGTVEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKIKTGYSTSADVLEKLAPYHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTKKVHTIFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSESDWLIFAADYSQIELRVLAHIAEDDNLMEAFRRDLDIHTKTAMDIFQVSEDEVIPNMRRQAKAVNFGIVYGISDYGLAQNLNISRKEAAEFIERYF Q SFPGVKRYMENIVQEAKQKGYVTILL R RRRYLPDITSRNFNVRSFAERMAMNIPIQGSAADIIKKAMIDLNARLKEERLQA A LLLQVHDELILEAPKEEMERLCRLVPEVMEQAVTLRVPLKVDY R YGSTWYDAK

SEQ ID NO: 4 contains three additional amino acid substitutions ascompared to SEQ ID NO:3: His281Ala, His273Arg, and Tyr477Phe. Thus SEQID NO: 4 contains a total of eight amino acid substitutions as comparedto the reference wild-type sequence of SEQ ID NO:1. The substitutedamino acids as compared to SEQ ID NO: 1 are underlined and in bold.

SEQ ID NO: 4 MAKMAFTLADRVTEEMLADKAALVVEVVEENYHDAPIVGIAVVNE R GRFFLRPETALADPQFVAWLGDETKKKSMFDSKRAAVALKWKGIELCGVSFDLLLAAYLLDPAQGVDDVAAAAKMKQYEAVRPDEAVYGKGAKRAVPDEPVLAEHLVRKAAAIWELERPFLDELRRNEQDRLLVELEQPLSSILAEMEFAGVKVDTKRLEQMGKELAEQLGTVEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPY R EIVENIL A YRQLGKLQSTYIEGLLKVVRPDTKKVHTIFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSESDWLIFAADYSQIELRVLAHIAEDDNLMEAFRRDLDIHTKTAMDIFQVSEDEVTPNMRRQAKAVNFGIVYGISDYGLAQNLNISRKEAAEFIERYF Q SFPGVKRYMENIVQEAKQKGYVTTLL R RRR F LPDITSRNFNVRSFAERMAMNTPIQGSAADIIKKAMIDLNARLKEERLQA A LLLQVHDELILEAPKEEMERLCRLVPEVMEQAVTLRVPLKVDY R YGSTWYDAK

The mutated nucleic acid sequences encoding SEQ ID NO: 2, SEQ ID NO: 3and SEQ ID NO: 4 were expressed in an E. coli host, and the modifiedproteins were purified using conventional techniques.

Example 2: Determination of Candidate Amino Acids for Modification in E.Coli SSB

The amino acid sequence of the single stranded DNA binding protein of Ecoli having the amino acid sequence of SEQ ID NO: 19 was analyzed usingPropKa.

(SEQ ID NO: 19)   1ASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLF  61GKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGA 121PAGGNIGGGQPQGGWGQPQQPQGGN

The calculated pKas of the amino acid residues having titratable sidechains are shown in Table 4.

Amino acid residues having calculated pKa values within the range ofabout pH 4.0 to about pH 10.0 are targeted for substitution. ModifiedSSB proteins comprising these substitutions are generated, expressed andpurified using conventional methods.

Example 3: Determination of Candidate Amino Acids for Modification inKOD DNA Polymerase

The amino acid sequence of the Therminator™ DNA polymerase (SEQ ID NO:5) is shown below:

(SEQ ID NO: 5) MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKFLGRPIEVWKLYFNHPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFDIETLYHEGEEFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKRFLRVVREKDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYKRNELAPNKPDERELARRRGGYAGGYVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADIDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEGFYVRGFFVIKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQIIRDLRDYKATGPHVAVAKRLAARGVKIRPGIVISYIVLKGSGRIGDRAIPADEFDPIKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK

The amino acid sequence of Therminator™ polymerase (SEQ ID NO: 5) wasanalyzed using PropKa. The calculated pKas of the amino acid residueshaving titratable side chains of the Therminator™ polymerase are shownin Table 5. Column 1 shows the amino acid residue; column 2 shows thepKa of the amino acid residue in the protein as calculated by PropKa;and column 3 shows the model pKa value for the amino acid in solution.

Amino acid residues having calculated pKa values within the range ofabout pH 4.0 to about pH 10.0 are targeted for substitution employed anyof the principles of selection and modification described herein.Nucleic acid sequences encoding the resulting modified polymerasescomprising these substitutions are synthesized, cloned, expressed andpurified using conventional genetic engineering methods.

Example 4: Determination of Candidate Amino Acids for Modification inKOD DNA Polymerase

The amino acid sequence of the KOD DNA polymerase (SEQ ID NO: 6) wasanalyzed using PropKa.

(SEQ ID NO: 6) MILDTDYITEDGKPVIRIFKKENGEFKIEYDRIFEPYFYALLKDDSAIEEVKKITAERHGTVVIVKRVEKVQKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDVVSTEREMIKRFLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFALGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWEIGENLERVARYSMEDAKVIYELGKEFLPMEAQLSRLIGQSLWDVSRSSIGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGGYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDILNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLDYRQRAIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDIDGFFATIPGADAETVKKKAMEFLKYINAKLPGALELEYEGFYKRGFFVIKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHEQIIRDLKDYKATGPHVAVAKRLAARGVKIRPGIVISYIVLKGSGRIGDRAIPFDEFDPIKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGT

The calculated pKas of the amino acid residues having titratable sidechains are shown in Table 6. Column 1 shows the amino acid residue;column 2 shows the pKa of the amino acid residue in the protein ascalculated by PropKa; and column 3 shows the model pKa value for theamino acid in solution.

Amino acid residues having calculated pKa values within the range ofabout pH 4.0 to about pH 10.0 are targeted for substitution. Modifiedpolymerases comprising these substitutions are generated, expressed andpurified using conventional methods.

Example 5: Determination of Candidate Amino Acids for Modification inB103 DNA Polymerase

The amino acid sequence of a B103-type polymerase, derived from thepublished sequence of the DNA polymerase of bacteriophage B103, is shownbelow as SEQ ID NO: 7.

(SEQ ID NO: 7)   1mprkmfscdf etttklddcr vwaygymeig nldnykigns ldefmqwvme iqadlyfhnl  61kfdgafivnw lehhgfkwsn eglpntynti iskmgqwymi dicfgykgkr klhtviydsl 121kklpfpvkki akdfqlpllk gdidyhaerp vgheitpeey eyikndieii araldiqfkg 181gldrmtagsd slkgfkdils tkkfnkvfpk lslpmdkeir rayrggftwl ndkykekeig 241egmvfdvnsl ypsqmysrpl pygapivfqg kyekdegypl yiqrirfefe lkegyiptiq 301ikknpffkgn eylknsgaep velyltnvdl eliqehyemy nveyidgfkf rektglfkef 361idkwtyvkth ekgakkqlak lmlnslygkf asnpdvtgkv pylkedgslg frvgdeeykd 421pvytpmgvfi tawarfttit aaqacydrii ycdtdsihlt gtevpeiikd ivdpkklgyw 481ahestfkrak ylrqktyiqd iyakevdgkl iecspdeatt tkfsvkcagm tdtikkkvtf 541dnfrvgfsst gkpkpvqvng gvvlvdsvft ik

Further details regarding the B103-type polymerase of SEQ ID NO: 7 canbe found in U.S. Patent Publication No. 20110014612.

Mutations to the B103-type DNA polymerase of SEQ ID NO: 7 to removepotential buffering side chains and thereby reduce the bufferingcapacity of the B103-type polymerase may include replacement of any oneor more histidine residues with arginine and/or replacement of any oneor more glutamic acid residues with alanine, as shown in Table 7.Mutations may further include replacement of any one or more cysteineresidues with serine, also as shown in Table 7.

Optionally, the modified B103-type polymerase can also include the aminoacid sequence of SEQ ID NO: 7 and further include the amino acidsubstitution D166A, which reduces 3′ to 5′ exonuclease activity. Thereduction or elimination of exonuclease activity can be helpful inapplications involving the detection of nucleotide incorporation, suchas ion-based nucleic acid sequencing.

The amino acid sequence of the B103-type DNA polymerase of SEQ ID NO: 7is highly homologous to the DNA polymerase of the bacteriophage Phi29,as shown in SEQ ID NO: 18.

(SEQ ID NO: 18)   1MKHMPRKMYS CAFETTTKVE DCRVWAYGYM NIEDHSEYKI GNSLDEFMAW VLKVQADLYF  61HNLKFAGAFI INWLERNGFK WSADGLPNTY NTIISRMGQW YMIDICLGYK GKRKIHTVIY 121DSLKKLPFPV KKIAKDFKLT VLKGDIDYHK ERPVGYKITP EEYAYIKNDI QIIAEALLIQ 181FKQGLDRMTA GSDSLKGFKD IITTKKFKKV FPTLSLGLDK EVRYAYRGGF TWLNDRFKEK 241EIGEGMVFDV NSLYPAQMYS RLLPYGEPIV FEGKYVWDED YPLHIQHIRC EFELKEGYIP 301TIQIKRSRFY KGNEYLKSSG GEIADLWLSN VDLELMKEHY DLYNVEYISG LKFKATTGLF 361KDFIDKWTYI KTTSEGAIKQ LAKLMLNSLY GKFASNPDVT GKVPYLKENG ALGFRLGEEE 421TKDPVYTPMG VFITAWARYT TITAAQACYD RIIYCDTDSI HLTGTEIPDV IKDIVDPKKL 481GYWAHESTFK RAKYLRQKTY IQDIYMKEVD GKLVEGSPDD YTDIKFSVKC AGMTDKIKKE 541VTFENFKVGF SRKMKPKPVQ VPGGVVLVDD TFTIK

The crystal structure of the Phi 29 DNA polymerase is known (Kamtekar etal., EMBO J. 25: 1335-1343 (2006). As the DNA polymerase of B103 islikely to fold into a similar structure as that of Phi29, the sequenceof the Phi29 DNA polymerase (SEQ ID NO:18) is analyzed by a program suchas PropKa to determine amino acid residues having pKas within the rangeof about pH 4.0 to about pH 10.0. Substitutions are then made to thecorresponding amino acids in the B103 DNA polymerase, as determinedusing the amino acid sequence alignment shown in FIG. 2.

Modified polymerases comprising any of the above substitutions aregenerated, expressed and purified using conventional methods.

Example 6: Assay for Non-Buffering Properties of Modified Proteins

The buffering properties of the modified Bst DNA polymerases of SEQ IDNO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 are evaluated by performing astandard titration as known in the art. The titration curves for themodified polymerases are compared to the titration curve for theunmodified protein having the amino acid sequence of SEQ ID NO: 1.Similar titrations are carried out for other modified proteins asdescribed in Examples 2-5.

Example 7: Use of a Modified Bst DNA Polymerase in an ExemplaryIon-Based DNA Sequencing Reaction

The modified Bst polymerase of SEQ ID NO: 2 was compared to theunmodified polymerase (SEQ ID NO: 1) in a sequencing reaction using thePersonal Genome Machine (PGM™) System (Applied Biosystems, Part Number4462921) The Personal Genome Machine™ System uses an array ofsemiconductor sensors to measure hydrogen ions generated when thepolymerase adds nucleotides to a DNA template.

The unmodified Bst DNA polymerase having the amino acid sequence of SEQID NO: 1 and the modified Bst DNA polymerase having the amino acidsequence of SEQ ID NO: 2 were used to sequence nucleic acid templatesusing an Ion Torrent PGM™ sequencer. Exemplary instructions forperforming ion-based sequencing using the Ion Torrent PGM™ sequencer canbe found in the User Guides provided with the Ion Torrent PGM™ 314Sequencing System (including the PGM™ 314 Library Preparation UserGuide, the PGM™ Template Preparation User Guide, and Ion PGM™ 315Sequencing Chip User Guide and the Ion Torrent PGM™ Sequencing UserGuide, all of which are incorporated by reference herein.

Sequencing was performed according to the following exemplary protocol:

Library Preparation

Briefly, the library preparation protocol was used to prepare a DNAlibrary having fragments with sizes having a distribution range of 50-60bp around a median size of 180-210 bp. Each library fragment is flankedby A and B adapters, allowing subsequent amplification.

The library was prepared essentially using the following steps:

DNA Fragmentation:

The DNA was sheared with the BioRuptor® Sonication System, essentiallyaccording to the protocol provided by the manufacturer.

An aliquot of the sonicated DNA was diluted and analyzed on aBioAnalyzer High Sensitivity DNA LabChip® or on an agarose gel toconfirm a fragment size range between ˜50-500 bp, with a peak around 200bp.

The fragmented DNA was end-repaired using the buffer and enzyme mixsupplied in the Ion Fragment Library Kit essentially using the followingsteps:

The end-repair reaction mix was prepared according to the followingTable and then incubated for 20 minutes at room temperature:

Component Volume Fragmented DNA (step 5.1.9) Y μL Nuclease-free Water158-Y μL 5X End Repair Buffer 40 μL End Repair Enzyme 2 μL Total 200

The DNA was purified with the Agencourt® AMPure® XP Kit.

Adapter Ligation:

The Amplification Adapters provided with the Ion Torrent PGM™ wereligated to the DNA using Ligase enzyme and the provided buffer. Thebuffer and ligase were mixed with the end-repaired DNA in nuclease freewater and let sit at room temperature for 30 minutes.

The DNA was then purified using the Agencourt® AMPure® XP Kit (BeckmanCoulter), essentially according to the protocol provided by themanufacturer.

The purified DNA was then size-selected using the Pippin Prep®instrument (SAGE Biosciences), essentially according to the protocolprovided by the manufacturer. When the separation was complete, thesize-selected DNA was recovered from the Elution Chamber and transferredto a new microcentrifuge tube.

The DNA was then purified using the Agencourt® AMPure® XP Kit (BeckmanCoulter), essentially according to the protocol provided by themanufacturer.

Nick-Translation and Amplification:

A nick-translation and amplification reaction mixture was prepared bymixing the DNA and primer mix with Platinum® PCR SuperMix High Fidelityand amplified on a thermocyling block using standard PCR reactionconditions.

The DNA was then purified using the Agencourt® AMPure® XP Kit (BeckmanCoulter), essentially according to the protocol provided by themanufacturer.

The library was then linked to washed Dynabeads M-270 Streptavidin beadsby incubating the washed beads with the size-selected and purified DNA,and incubating at room temperature with shaking at 8-10 rpm for 20minutes.

The bead-linked library was washed twice in buffer, then resuspended inAlkali Solution including 0.125N NaOH. The supernatant was collected,neutralized and purified using the sample using the QIAGEN MinEluteColumn essentially according to the protocol of the manufacturer.

Quality assessment and library quantification was performed theRiboGreen® RNA quantitation kit or the BioAnalyzer™ RNA 6000 PicoLabChip® essentially according to the protocol of the manufacturer.

Template Preparation

Nucleic acid templates were amplified onto hydrogel matrices (“IonSpheres”, prepared essentially as described in), essentially accordingto the following protocol:

Determination of Optimal Library Concentration for emPCR

The concentration of the template library was estimated, and thetemplate DNA was diluted to an appropriate concentration. Emulsion PCRwas then performed by mixing the following components:

Reagent Volume (μL) Nuclease-free water 344 AmpMix 105 MgCl₂ Solution105 dNTPs 105 Primers Mix 105 TIPP (NEB) 2 Polymerase 126 Ion Sphere ™Particles 140 Library 18 Total 1050

The amplification mix was then transferred into an oil phase withshaking. The emulsion was then subjected to PCR amplification usingstandard thermocycling conditions.

The Ion Sphere Particles including amplified nucleic acid was thenrecovered by extracting the emulsion with butanol and hexane, and thenwashing the recovered particles in buffer.

Template-positive Ion Sphere™ particles were enriched by washing thespheres with denaturation solution, incubating with an enrichment primerand washed Dynabeads® MyOne™ Streptavidin C1 beads. The enrichedparticles were then separated from the Dynabeads® MyOne™ Streptavidin C1beads using appropriate washes in alkali solution including NaOH.

Sequencing

Amplified nucleic acid templates bound to hydrogel matrices and preparedessentially according to the foregoing protocol were sequencing in theIon Torrent PGM™ sequencing using the sequencing primer supplied withthe sequencing kit. Essentially, the amplified templates on the hydrogelmatrices were suspended in sequencing buffer and pipetted into the IonTorrent 314 Sequencing Chip, which was placed in the Ion Torrent PGM™Sequencer. The sequencing run was initiated and performed by the PGM™sequencer. The reagent conditions for sequencing were 6.3 mM NaCl₂, 13mM MgCl₂, 0.1% Triton X-100, adjusted to pH 7.5 with NaOH. 1 μl of a 59μM solution of the polymerase was added to 10M beads per 314 chipsequencing. The nucleotide concentration was 50 uM.

Data Analysis

This section provides an overview of the software modules and conceptsapplied at various stages of the data processing of the signals gatheredduring the sequencing reaction. The Torrent Server Analysis Pipeline,the “Pipeline”, processes raw acquisition data from a PGM run, andoutputs base calls in both SFF and FASTQ file formats. The TorrentBrowser provides a web interface to the process, including many metrics,graphs, and reporting features derived from the Pipeline results.

During a PGM™ run, for each nucleotide flow, one acquisition file isgenerated. Each acquisition file contains the raw signal measurement ineach well of the chip for the given nucleotide flow. So each acquisitionflow, for a 314 chip, contains roughly 1.5 million separateincorporation events. A series of such acquisition files then representsthe roughly 1.5 million possible reads. The analysis pipeline convertsthese raw signal measurements into incorporation measures, andultimately into base calls for each read. The raw measurements are thesystem's conversion of the raw pH value in each well into a voltageconverted into a digital representation of that voltage. Thismeasurement over the entire chip occurs many times per second.

The following passages describe briefly the high level modules withinthe analysis pipeline. Each module accepts specific inputs and producesspecific outputs, described in more detail, below.

DAT Processing

The DAT processing module deals directly with raw acquisition files(acq_*.dat).

DAT processing performs the following functions:

Raw Acquisition Data Loading into Memory.

This includes decompressing the data files. Data is compressed when itis streamed off the PGM™. An optional dynamic frame rate compressionmode whereby various portions of the incorporation event throughout thenucleotide flow duration are captured at different frame rates. Thevariable frame rate allows biologically specific events to be capturedwith high resolution, while at the same time allowing the overall filesize to be decreased by allowing multiple frames to be averaged whereappropriate. Alternatively, a compression approach may use akeyframe/delta compression technique whereby an initial value is stored,followed by the changes in that initial value, rather than storing theactual value each time. This results in a nearly 2× reduction in filesize.

Raw Measurement Offset Corrections:

Raw acquisition data are stored using the values output by the chip.Each well has its own reference value, and to compare well to well, thetwo wells may use a common reference. The offset correction takes theaverage of the first few frames within each acquisition file andsubtracts that value from each well, thus allowing well measurements tohave a common reference value.

Pinned Well Identification:

Due to the nature of the chip output voltages, a range of values existsfor any given chip. The PGM instrument calibration code brings themajority of wells within range of the hardware's analog to digitalconverters. The output is a distribution of values centered around thecenter voltage. Wells that reside outside a selected distribution areconsidered ‘pinned’ (functional wells outside of the range of the ADC).These pinned wells typically represent less than one percent of thetotal available wells.

Excluded Wells:

Various flow cell configurations often make tradeoffs on flow velocityprofiles and chip coverage areas. For the 314 chip, for example, apercentage of the wells are covered by the flow cell and are notfluidically addressable. A mask is loaded, per chip type, to mark thosewells as excluded so they do not complicate down-stream processing ofthe chip.

Classification

Classification is the process of determining the contents of each wellon the chip. The following describes the classification decision tree: Awell can either be empty, or contain a particle. For wells containing aparticle, the process additionally determines if that particle is: (a) Alibrary particle; (b) A test fragment (control) particle; (c) A dudparticle (a particle that does not produce a sufficiently strongsequence signal); or (d) An ambiguous particle (a particle where itappears to the software as both a test fragment and a library fragment).It is important to note at this point that classification is not simplyprocessing the entire chip as one group. As will be mentioned for otherpipeline modules, the chip is processed in smaller regions. A 314 chip,for example, is segmented into 50×50 well regions, resulting in about625 total regions. This allows processing many small regions in paralleland taking advantage of multi-core/multi-process compute nodes that havesuch capabilities. More importantly, regions in the chip are processedthat contain fluidically similar wells, where smaller regions tend to berelatively homogeneous and allow comparison of wells to each otherwithin a region.

Default Sequencing Keys and Flow Order

The next part of the classification module involves identifying particlewells as test fragments (TF) or library fragments. To understand thispart of the algorithm, the concept of ‘keys’ and flow order must firstbe understood. Table 8, below shows the default library and TFsequencing keys, in both base-space and in flow-space for the givendefault flow order TACG.

TABLE 8 Base-space Flow order TACG vector Library Key TCAG 1010010X TFKey ATCG 0100101X

The ‘X’ in the vector, above, indicates that the value is at least aone, but could in fact be higher because the next base after the keycould also match, thus creating a longer homopolymer.

Separation

Once the particles have been identified within the wells, separation isthen performed. In this step, the two sequencing keys are used tocompare each read. The well is determined to contain a bead; the flowsare converted into a sequence and tested against each key. Because thetest is performed against both keys, the number of flows needed tosequence each key is determined, and the lower number of the two isused. Adding many more bases to one of the two keys will not alwaysresult in better separation since the shorter of the two drives thenumber of flows ultimately used.

With the default keys and flow order, above, there are two vectors:

TACGTACG

1010010

0100101

Here, valid comparison nucleotide pairs are then searched for. Thismeans that to compare two sequences, the following rules need to besatisfied: (1) That there is a 0-mer and a 1-mer event for eachnucleotide in the key; and (2) That there is the 1-mer from one keyfiring during the 0-mer event in the other nucleotide.

When both rules are satisfied for a given nucleotide, there is aseparator ‘event’ to be used. The more ‘events’ there are, the betterthe two keys can be separated. In other words, the nucleotide pairs areorthogonal in flow-space for the two keys, as in the bold verticalhighlighted pairs:

‘T’ nuc satisfies our rules:

1010010

0100101

Above, for the ‘T’ nuc flow, the library key incorporates on the firstflow, and has a 0-mer on the second ‘T’ flow, whereas the TF key has theopposite.

A similar case is observed for the ‘A’ nucleotide, and the ‘C’nucleotide. The ‘G’ nucleotide cannot be used because there is no 1-merevent observed until the 8th flow. That flow cannot be included becausethat ‘G’ in the library can be part of a larger homopolymer stretch.Thus, it cannot be guaranteed that there is exactly a 1-mer in thatflow. Further, the TF key does not have a 1-mer in the first ‘G’ flow,but has a 0-mer ‘G’ in the first ‘G’ flow which is the same as thelibrary key.

‘A’ nucleotide satisfies these rules:

1010010

0100101

‘C’ nucleotide satisfies these rules:

1010010

0100101

Mask

The output of the classification module is a mask object (and file asbfmask.bin) that contains bit flags for each well, indicating thecontents of each well.

Signal Processing

The signal processing module focuses on wells containing particles,which is now using the mask information in addition to the rawacquisition data. A signal is measured in a well during an incorporationevent. The signal has additional components, referred to as ‘thebackground’. This background part of the signal is present during eachflow and can vary over time, across the chip, and during an acquisition.The signal processing problem can be thought of as having two parts. Thefirst part involves deriving the background signal that would have beenmeasured in a given well, had no incorporation occurred. The second partinvolves subtracting (or fitting) the background signal and examining(or fitting to) the remaining signal. The output of the incorporationfitting model produces the estimate of the incorporation at eachnucleotide flow for each well.

At this point, the output from the analysis pipeline is a rawincorporation signal measure per well, per flow, stored as a 1.wellsfile.

Basecalling

The basecaller module performs signal normalizations, phase and droopestimations, signal corrections, and declares bases for each flow ofeach well. It outputs non-incorporation events in addition toincorporation events. The SFF output file stores all such calls.

Normalization

Normalization is performed on each read. Normalization is a way of usingthe known expected 1-mer signals produced by sequencing through the key,and using those signals to establish the 1-mer average signal. This isinitially a process using the raw measurements from the signalprocessing module. As the basecaller processes each well, more bases areaccurately determined and additional measurements can then be used tore-normalize the raw measured signals to gain higher confidence, ahigher signal-to-noise ratio (SNR), in the normalization of each well.

Droop Estimation

Observed signal droop can be attributed to DNA polymerase loss that canoccur during a sequencing run. Typically, such loss is experienced onlyduring incorporation events, and typical values are in the 0.1 to 0.2%range over the course of a run. By averaging groups of reads in a regiontogether and averaging their measured signals (after normalization), anexponential can be fit to the resulting curve, and the rate of signalloss over time is extracted. Thus, an estimate of the polymerase lossduring incorporation events can be determined.

Phase Estimation

Once the droop has been established, it is used in the phase model as aconstant for each read. Droop estimates can still vary across the chip,but is assumed fixed for each processed region. The model then fits thecarry-forward and incomplete extension parameters for each read, over alimited number of flows and excluding certain flows. The output fromthis fit is an estimate of the carry-forward (CF) and incompleteextension (IE) for each well. The values are averaged over small regionsto reduce errors and noise in the fit. The output CF and IE values areused as inputs to the solver.

Solver

The solver part of the base caller applies the phase and droop estimatesto the normalized signals and makes predictions of the likely measuredvalues for each flow for probable incorporation events. By comparing theactual measured value to the list of predicted values, the best fitprediction at each flow is then used as the base call for that flow. So,a 0-mer, 1-mer, 2-mer, etc. can be predicted at each flow. This processcontinues over the entire read. At the end of one pass, a good estimateof all bases for that read have now been made. The solver then iteratesover the read again, applying the same phase and droop estimates at eachflow, and refines the base calls. This refining is beneficial sinceknowledge of future bases on future flows can now be included in themodel to more accurately account for carry-forward effects.

Read Filtering and Trimming

Read filtering involves generation of various quality metrics for eachbase call, quality metrics for each read, and using those metrics tofilter out low quality reads and trim reads back to acceptable qualitylevels. Additionally, adapter trimming is performed on the ends of eachread.

Filtering

Filtering reads involves calculating an overall quality metricrepresenting the base caller's ability to accurately correct and basecall the raw signals. Reads that have calculated low quality are notwritten to the SFF file. Low quality reads are typically mixed reads orvery low copy-count reads that produce low quality sequence such thatthey do not fit the expected incorporation model.

Briefly, the Ion Torrent PGM™ system applies some quality checks tosequence reads before writing them out. Reads are tested to see if theyare possibly the result of mixed DNA templates on a single Ion Sphereparticle in a given well, or if they are generally of low signalquality. The 3′ ends of reads are scanned for matches to the adaptersequence and for regions of trailing low quality. Only the high-qualityportions of reads passing through these checks are written out to theSFF and FASTQ files.

When processing data from the Personal Genome Machine (PGM™), theidentification and removal of low-quality or uncertain base calls is animportant consideration for delivering accurate results. In the TorrentSuite analysis software, low-quality bases are removed from the outputby: (a) Filtering out entire reads; (b) Trimming low-quality 3′ ends ofreads.

The operations described in the following passages are applied aspost-processing operations after the initial base calls have beenestimated.

Read Filtering

The goal of read filtering is the removal of reads that are estimated tocomprise low-quality base calls. The kinds of read filters include: (1)A read filter targeted at removal of reads that are derived from wellswith non-clonal DNA template populations (i.e. mixtures of different DNAtemplates); (2) A read filter targeted at reads that are generally apoor fit to the basecalling model's expectations for high-quality data.

Removal of Mixed-Template Reads

Sometimes the DNA template that ends up being amplified on the IonSphere particle is derived from multiple different input DNA templates.Possible ways in which this can occur include the presence of two ormore distinct DNA fragments in the vesicle at the start of the emulsionPCR stage, or the collapsing together of different emulsion vesicles.

In the usual scenario, each particle gets a single DNA template speciesamplified onto it, in which case approximately half of the nucleotideflows should result in a positive incorporation (assuming a 4-base flowcycle and uniform and random nucleotide content in the genome). When aparticle contains multiple different DNA templates, the number of flowsin which a positive incorporation signal can be expected increasessubstantially.

An effective way to identify possible mixed template reads is to searchfor reads in which an unusually large proportion of flows are estimatedto have a nucleotide incorporation event. As of version 1.1.0 of theTorrent Suite software, the percentage of positive flows (PPF) isevaluated over the first 60 flows (including the key flows). If the PPFvalue is greater than 60%, the read is excluded before writing out theSFF and FASTQ results. In runs with fewer than 60 flows, the PPF isevaluated over however many flows are available.

One implication of this PPF filter is that certain sequences may not becalled by the software. For example, a long sequence that is in exactlythe flow order TACG would be expected to have a positive incorporationon every flow and as a result would be filtered out. However in practicesequences like this are rare and the benefits of excluding low-qualityreads resulting from mixed templates are favored over excluding a smallproportion of genuine reads.

One exception to this approach pertains to test fragments, some of whichare designed with sequences that do not typically occur naturally andthat have a large expected PPF value. By default, the software does notapply the PPF filter to test fragment reads.

Removal of Lower Quality Reads

The basecalling model has certain expectations about the signaldistribution for well-behaved reads. Once the amount of incompleteextension (IE) and carry forward (CF) phasing effects have beenestimated there are certain expectations for how signals in neighboringflows should be elevated or depressed. For example, when there ispositive IE in effect, a large homopolymer run should result in adepressed signal value in the flow corresponding to the homopolymer andan elevated signal value in the next flow of the same nucleotide.

As part of the basecalling model the observed signal value is comparedwith what the basecalling model predicted in each flow for each read.The difference between these two quantities (observed value minuspredicted value) is referred to as the flow residual for the well andflow in question. In general, a high-quality read which iswell-described by the basecalling model and the DNA sequence that itestimates should have low residual values.

The median absolute value of the residual over the initial flows istracked for each read as a measure of the agreement between observeddata and basecalling model. If the median absolute residual is greaterthan a certain threshold, then the read is considered to be untrustedand it is not written to the SFF and FASTQ output files.

As of version 1.1.0 of the Torrent Suite software the median absoluteresidual is computed over the first 60 flows (including key flows) andthe threshold above which a read is rejected is 0.13.

The median absolute residual filter is applied for both library andtest-fragment reads.

Trimming

The goal of read trimming is the removal of undesired base calls at the3′ end of a read. Such undesired base calls come from: (a) High-qualitybase calls on reads that have read all the way through the libraryinsert into the B-adapter; and (b) Low-quality base calls at the 3′ endof the read.

Two filters are applied to trim reads, one targeted at each of the twocategories of undesirable 3′ bases. Each filter is applied separatelyand the read length is taken as the shorter of the two. If the resultingtrimmed length is shorter than the minimum read length, the read isfiltered out entirely, otherwise the read is written to the SFF and theFASTQ files.

As long as trimming does not reduce the read length below the minimumdesignated allowable length (four bases as of version 1.1.0 of thesoftware), the flow values and base calls for the entire read arewritten into the SFF, regardless of the trimmed length. The trimminginformation is represented in the designated fields in the SFF file.

Removal of Adapter Sequence

Trailing adapter sequence is trimmed out by searching the read forcandidate matches to the known adapter sequence in flow-space.

The basecaller works by reversing the effects of CF and IE, producing aphase-corrected ionogram that is stored in the SFF file. If a readextends into the B-adapter, the 3′ end of this phase-corrected ionogramwill exhibit a pattern that is characteristic of the adapter sequence.

Adapter trimming is accomplished by testing each position in thephase-corrected ionogram to see how well it matches the pattern expectedfor the adapter. The test computes the Euclidean distance between thephase-corrected ionogram at the tested position and the known ionogramfor the adapter. If this distance falls below a fixed threshold, thecorresponding position (translated from flow-space back to read-space)is recorded as the adapter trim location; otherwise, the read isconsidered not to have a match to the adapter. As of version 1.1.0 ofthe Torrent Suite software, the threshold used is a Euclidean distanceof 5.

Removal of Lower-Quality 3′ Ends

The distribution of quality scores within Ion Torrent reads is such thatthe highest quality calls tend to occur at the start of the read wherephase errors are smallest in magnitude. For reads that run intolow-quality bases before reaching the end of the template, it is helpfulto trim away the lower-quality calls at the 3′ end. The quality trimmingis performed using the per-base quality scores (described further below)

The approach is to scan along the bases of the read computing a movingaverage in a fixed-length window, and to set the read trim point to justbefore the earliest (5′-most) base at which the moving average of theper-base quality scores drops below some fixed threshold.

As of version 1.1.0 of the Torrent Suite software the window size is setto 30 bases and the threshold below which the trimming occurs is aquality score of nine.

Quality and Adapter Trimming

Using processing algorithms, a read can be trimmed back until anacceptable level of quality persists for the entire read. A read is alsoexamined to determine if the B-Primer adapter sequence or a portionthereof can be identified with high confidence within the read. If amatching sequence is found, the read is trimmed to the base immediatelyprior to the start of the adapter.

Trimmed SFF File

The output of this stage is an SFF file that contains only the filteredreads and, for each read, the adapter and quality trimming markers areset appropriately. It is this file that is used to generate the FASTQfile.

Alignment QC

The alignment QC stage involves alignment of reads produced by thepipeline to a reference genome, and extracting metrics from thosealignments. Currently, the TMAP aligner is used to align reads. Asinputs to the aligner, some or all of the reads produced in the pipelineare used, along with the reference genome and index files. The output isa SAM file. This output SAM file is processed to extract various qualitymetrics, including estimates of Q17 or Q20 bases, estimates of number ofreads at or above 100Q17. The results of the Alignment QC module areviewable using the Torrent Browser web interface in the Reports summarypage. The key output component is the SAM file.

The following table shows the files output at each stage or module,along with their expected size for a typical 314 chip library run:

TABLE 9 Module/Stage File type Typical 314 run size DAT Processing *.dat53 GB Classification bfmask.bin 3 MB Signal Processing 1.wells 1237 MBBasecalling SFF 515 MB Read Filtering and Trimming SFF, FASTQ 515 MB,122 MB Alignment QC SAM 183 MB

Per-Base Quality Scoring

Per-base quality scores are assigned to each read, and written to theSFF file along with the read itself. The per-base quality scores areassigned by calculating various metrics for the read and using thosemetrics as lookup indexes into a pre-defined quality lookup tableestablished through prior system training. The metrics often involveestimates of accuracy at the current base, flow, and earlier or laterbases or flows for each read.

The Ion Torrent per base quality score system uses a phred-like lookuptable to predict quality. The lookup table is generated in training froma representative data set and used to predict the quality of PGM data ona per base basis. Quality scores are published in the SFF, FASTQ and SAMfiles.

The quality score system includes: (1) A phred-like per base lookuptable; and (2) A training system to generate the lookup table.

From read flow values, several quality predictors are calculated foreach base in the read. These predictors are used as part of an index tolookup an appropriate quality score for the base in the phred lookuptable. See, e.g., Brockman et al. (2008): “Quality scores and SNPdetection in sequencing-by-synthesis systems.” Genome Res. 18: 763-770;Ewing B, Hillier L, Wendl M C, Green P. (1998): “Base-calling ofautomated sequencer traces using phred. I. Accuracy assessment.” GenomeRes. 8(3):175-185; Ewing B, Green P. (1998): “Base-calling of automatedsequencer traces using phred. II. Error probabilities.” Genome Res.8(3):186-194.

A training system uses the quality predictors to derive the lookuptable.

Training System

To generate a lookup table a representative data set is selected as atraining set. The training system uses the following six predictors tocapture the majority of features that correlate with and indicatequality:

TABLE 10 Six Predictors used to capture features correlating with andindicating quality P1 Base position within the read from the start ofthe sequence. P2 Local noise in the immediate neighborhood (plus/minus 1base) of the given base: P2 = max [abs(flowval[base]) −round(flowval[base])]. P3 Read noise as a peak-normalized expression ofthe mean and standard deviation of all 0-mers and 1-mers in the read: P3= − (m1 − m0 − s1 − s0)/m1. P4 Multiple incorporations: In case ofmultiple incorporations of the same nucleotide in one flow, the lastbase in the incorporation order is assigned a value equivalent to thetotal number of incorporations. All other bases in the sequence of themultiple incorporations are assigned the value 1. P5 Phase error: Thenumber of incorporations of the same nucleotide in the previous flow. P6Environment noise in the larger neighborhood (plus/minus 10 bases) ofthe given base: P6 = max [abs(flowval[base]) − round(flowval[base])].

The quality predictors are calculated from the flow values for eachbase. Together with TMAP-aligned reads as input, the system establishesthese six predictors, along with whether or not the base was calledcorrectly. This vector of one “match” boolean plus six predictors foreach base is used as the input into a training system. The predictorsare binned, and an extensive list of all combinations with theirempirical quality score is recorded. An algorithm is used to summarizepredictor values that correspond to the same quality and to select arepresentative subset of these combinations.

The output of the training and selection algorithm is a lookup table inwhich each predictor is used as part of an index into the table to lookup the phred-like per base quality score at that location.

The Lookup Table

The lookup table is used to assign per base quality scores independentof alignment. The six quality predictors are calculated for each basesequenced by the PGM. The corresponding per base quality is located inthe lookup table by finding the first line for which all six calculatedpredictors are less than or equal to the predictor values in the table.The lookup quality is read from the same line. This process occursautomatically as part of the standard analysis.

Pipeline Implementation

The Ion Torrent pipeline processes the signal from each pixel on thechip, applying corrections for background, noise, and phase effects, toproduce basecalls.

After bases are called, a separate class, PerBaseQual, is called fromwithin Analysis.cpp to calculate the quality predictors for each baseand assign a quality from the phred table.

The per base quality, along with all other read information is writtento the Standard Flowgram Format (SFF) file using WriteData method fromthe sff class. The positive integer quality scores per base are listedas the last data line for each read in the SFF file, under the Qualityscores heading. The quality scores are on a phred −10*log_10(error rate)scale.

In addition to the SFF file, Ion Torrent also saves the results in FASTQand Sequence Alignment/Map (SAM) files:

TABLE 11 SFF file The positive integer per base quality scores are underthe ‘Quality Scores’ heading. FASTQ The per base quality scores from 0to 93 are represented by file ASCII characters 33-126. SAM file The perbase quality scores are reported in the QUAL field in the form of ASCIIcharacters 33-126.

Results

The following Analysis Reports are generated and provided by the IonTorrent Browser: (1) Library Summary; (2) Test Fragment Summary; (3) IonSphere™ Particle Summary; (4) Report Information; and (5) File Links.

Files

SFF-formatted or FASTQ-formatted files that contain all library readsfor a specific run are generated and can be downloaded from the IonTorrent Browser, including the following files:

(1) Library Sequence (SFF) files: These files include Standard FlowgramFormat (SFF)-formatted files and contain “flow space” data. The data areorganized on a per flow basis, and contain information about bothnucleotide flows that resulted in base incorporation and those that didnot.

(2) Library Sequence (FASTQ) files: These files include FASTQ-formattedfile, which is organized in a per base basis, including quality scores.The reads contained in the file are unaligned reads.

(3) Library Alignments (SAM): These are Sequence Alignment/Map (SAM)files containing data that are already aligned.

(4) Test Fragments (SFF): These files include Standard Flowgram Format(SFF) data for Test Fragments only.

Analysis Log File: These files include a detailed log file useful fortroubleshooting

The following sequencing information is included in the SFF and theFASTQ files:

Read Filtering

Reads that meet the following criteria are reported in the SFF and FASTQfiles: The well associated with the read is “positive” for an IonSphere.

The well produced a strong signal (greater than 21 counts) across eachof the first three key nucleotides. The read contains a minimum of 8bases.

The key of the read is an exact match following basecalling, whichdefines a “keypass read.”

The read has between 40% and 60% of its flows registering as positive.Reads with greater than 60% positive flows are likely to be mixed reads.

The read fits the expected CAFIE model across the first 60 flows.

Read Trimming

Each reported read is subject to removing lower quality bases accordingto the following rules: The first four bases of the 5′ end that containthe key are removed.

No quality trimming occurs at the 5′ end.

At the 3′ end, if sequencing occurs through the insert to the B adapter,the B adapter is removed.

The 3′ end of the read is trimmed to the point where the average qualityscore is at least Q9 across a moving 30 bp window.

The FASTQ file only contains filtered and trimmed data. However, the SFFfile contains all data but has embedded annotation information thatdefines the filtered and trimmed regions.

ION Sphere™ Particle Identification Summary

This section of the report is available before the Library Summary orTest Fragment Summary sections are available to provide a quickdetermination of whether or not the analysis should be permitted tocontinue. The data generated in this section of the report are created“early” in the analysis process, before base calling, and are intendedto be a coarse, initial assessment of run performance. The well data aresubject to more stringent analysis in later stages of the analysis.

The report displays the following information:

Wells with Ion Sphere Particles:

Number of wells that were determined to be “positive” for the presenceof an ION Sphere particle within the well.

Live Ion Sphere Particles:

Number of wells that contained an ION Sphere particle with a strongsignal associated with the library or Test Fragment key. This value isthe sum of the following categories: (1) Test Fragment; (2) library

Test Fragment Ion Sphere Particles:

Number of Live ION Sphere particles with key signal that was identicalto the Test Fragment key signal.

Library Ion Sphere Particles:

Predicted number of Live ION Sphere particles that have a key signalidentical to the library key signal.

Template-Positive Ion Sphere Particles:

The estimated percentage of ION Sphere particles that contain sufficientDNA for sequencing. The ratio is approximately live library ION Sphereparticles over the total number of particles, less Test Fragment beadsand lower quality particles.

Library Key:

Four-base nucleotide sequence that identifies a read as a library read.This sequence is removed from the reported read in the SFF/FASTQ file.

Verified Ion Sphere Library Particles:

Number of ION Sphere particles where the four bases of the library keywere confirmed by the base calling algorithm. This is the number ofreads reported in the SFF & FASTQ files.

Library Summary

The Library Summary section of the Analysis Report contains performancemetrics for reads that contain the library key as the first four bases.

Performance is measured based on either predicted quality, or quality asmeasured following alignment.

Predicted Quality

The Q20 length value attributed to a read is an estimate of the readlength at which the predicted total error rate in the read willcorrespond to a Phred-scale quality score of 20. The Phred scale isdefined as 10×log 10(error probability), so Q20 corresponds to an errorrate of 1% and Q17 corresponds to an error rate of 2%.

The Q20 length is determined by looking at the per-base quality scoresto estimate the total read error rate at every position in the read. Forexample, if the first base had a predicted quality score of 20 and thesecond base had a predicted quality score of 17, then the estimatedtotal read error for the first two bases would be 0.5×1%+0.5×2%=1.5%.This total read error rate is evaluated at every position in the read,after which the maximal position at which the total read error rate is1% or less is identified. This position defines the Q20 length of theread.

The Q20 length is derived entirely from the predicted per-base qualityscores. The predicted quality scores are somewhat conservative and incases where alignment to a reference sequence is also possible userswill generally find that the predicted Q17 lengths tend on average to beshorter than the corresponding AQ17 lengths which are based on theactual as opposed to predicted errors (see the following section onAQ20/AQ17).

Quality Following Alignment

Alignment of reads can be a useful process to assess the quality of boththe sequencing reaction and the quality of the underlying library, wherea reference is available. Reads are aligned to a reference genome. Anydiscrepancy in alignment to a reference (whether biological ortechnical, meaning a real variant or a sequencing error) is listed as anerror. Alignment performance metrics are reported depending on how manymisaligned bases are permitted. Torrent Suite reports alignmentperformance at two quality levels: (1) AQ17; and (2) AQ20

The AQ17 length of a read is defined very similarly to the Q17 length(see above), the difference is that instead of using predicted per-basequality scores to estimate error rates we use the actual observed errorsbased on alignment.

The underlying assumption is that the reference to which the read isaligned represents the true sequence that we should have seen. Suitablecaution should be taken when interpreting AQ17 values in situationswhere the sample sequenced has substantial differences relative to thereference used—for example when working with alignments to a rough draftgenome, or with samples that are expected to have high mutation ratesrelative to the reference used. In such situations the AQ17 lengthsmight be short even when sequencing quality is excellent.

The AQ20 length is computed as follows: Every base in the read isclassified as being correct or incorrect according to the alignment tothe reference. At every position in the read, the total error rate iscomputed up to and including that position. The greatest position atwhich the error rate is 1% or less is identified, and that positiondefines the AQ20 length.

For example, if a 100 bp read consists of 80 perfect bases followed by 2errors and then 18 more perfect bases then the total error rate atposition 80 would be 0%, at position 81 it would be 1.2% (1/81), atposition 82 it would be 2.4%, and so on up to position 100 where itwould be 2% (2/100). The greatest length at which the error rate is 1%or less would be 80 and the greatest length at which the error rate is2% or less is 100, so the AQ20 and AQ17 lengths are 80 and 100 basesrespectively.

Alignment: Nucleic acid sequence information obtained using the IonTorrent PGM™ system is aligned with known or reference sequences usingstandard methods. Methods of aligning sequences are well known in theart and there are many alignment algorithms available within themarketplace. Alignment algorithms are also embedded in many of thecommercial software tools that are widely available. We also encourageyou to experiment with these tools. In some embodiments, alignmentwithin Torrent Browser is performed using TMAP. The precursor to TMAP,BFAST, is based on the ideas in the following publications: Homer N,Merriman B, Nelson S F., “BFAST: An alignment tool for large scalegenome resequencing.” PMID: 19907642, PLoS ONE. 2009 4(11): e7767,http://dx.doi.org/10.1371/journal.ponc.0007767; Homer N, Merriman B,Nelson S F., “Local alignment of two-base encoded DNA sequence.” BMCBioinformatics. 2009 Jun. 9; 10(1):175. PMID: 19508732,http://dx.doi.org/10.1186/1471-2105-10-175.

Library Summary Report

Performance, measured based on either predicted quality or quality asmeasured following alignment, is shown in the following sections of theSummary Report: (A) Based on Predicted Per-Base QualityScores—Independent of Alignment; (B) Reference Genome Information; and(C) Based on Full Library Alignment

(A) Based on Predicted Per-Base Quality Scores—Independent of Alignment

This section of the Library Report shows performance measured based onpredicted quality.

Total number of bases [Mbp]: Number of filtered and trimmed million basepairs reported in the SFF and FASTQ files.

In predicted Q17 read stretches [Mbp]: Number of filtered and trimmedmillion base pairs contained in read segments that extend from the readstart to the 3′-most base at which the total read accuracy is 98% orgreater (Q17).

In predicted Q20 read stretches [Mbp]: Number of filtered and trimmedmillion base pairs contained in read segments that extend from the readstart to the 3′-most base at which the total read accuracy is 99% orgreater (Q20).

Total number of reads: Total number of filtered and trimmed readsindependent of length reported in the SFF and FASTQ files.

At least 50 bp long: Total number of reads at 50 base pairs or longer.

At least 100 bp long: Total number of reads at 100 base pairs or longer.

Mean Length [bp]: Average length, in base pairs, of all filtered andtrimmed library reads reported in the SFF and FASTQ files.

Longest Read [bp]: Maximum length, in base pairs, of all filtered andtrimmed library reads either reported in the file.

The Read Length Histogram: This is a histogram of the trimmed lengths ofall reads present in the SFF file.

The Consensus Key 1-Mer: This graph shows the strength of the signalfrom the first three one-mer bases of the library key. This graphrepresents consensus signal measurement of release of H⁺ ions duringnucleotide incorporation. The y-axis shows signal strength, measured inCounts, which is an arbitrary but consistent unit of measure. The x-axisshows time as nucleotide flows over the chip.

There is a known key that exists at the start of every library read.Typically, three bases are shown of the 4-mer key. Note that the graphis displayed in “flow order” rather than “base order.” For example, thefour base library key is typically TCAG for nucleotides one throughfour. However, the graph displays that TCAG as TAC which represents theflow order of the nucleotides. Negative flows are not displayed.Although the library key is 4 bases long, only three bases are displayedin the key one-mer graph, because the last base of the library readisn't informative for quality purposes because that flow can containlibrary information in addition to key information. The next base afterthe last key base is the first library base. This base varies dependingon the library fragment. If the last key base is a G and the firstlibrary base is a G, then both of these are incorporated in the sameflow resulting in a signal roughly 2× of a one-mer. Thus the one-mer keypass graph only contains n-1 flows for an n-mer library key.

Test Fragment Summary

The Test Fragment Summary section of the Analysis Report providesinformation about the performance of each Test Fragment included in theexperiment (run).

The report displays a summary of the data from the specific TestFragment, for every Test Fragment found within the specific PGM™ run.

The following Quality metrics are displayed, for each Test Fragment, intabular form under the heading Test Fragment—<TestFragmentName>, asshown in the following figure:

TF Name: Test Fragment name, as defined in the Templates tab of TorrentBrowser

TF Seq: Test Fragment sequence.

Num: Number of filtered & trimmed reads identified for this TestFragment.

Avg Q17: Average read length with Q17, or better, for this TestFragment.

50AQ17: Number of reads for this Test Fragment with a minimum of 50 basepairs in length and an error rate of 1 in 50, PHRED-like 17, or better.Quality is based on alignment, not predicted quality.

AQ17 Read Lengths:

The Read Lengths graph is a histogram of read lengths, in bp, that havea Phred-like score of 17 or better (1 error every 50 bp). Distributionsthat are skewed to the right are ideal, showing longer read lengths(remember, Test Fragments are of a discrete length). Furthermore, it isvery likely that the sequence can extend all the way through the TestFragment provided enough cycles were run. Consequently, the histogramcan only display a maximum size based on the length of the Test Fragmentused.

Average Corrected Ionogram:

The Average Corrected Ionogram graphs the average corrected Ionogram forthis Test Fragment. The x-axis is in flow space and the y-axis shows thesignal intensity. A unit of one indicates that there is only a single“base” (i.e., nucleotide) incorporated in a particular flow space, whilea unit of two indicates that there are two “bases” (i.e., nucleotides)incorporated of that type in the particular flow space.

The following parameters are also reported, which contain the followinginformation about a particular sequencing run:

TABLE 12 Lib Key Signal Strength of the signal associated with thelibrary key. Q17 Bases Number of base pairs of sequence in predicted Q17stretches. 100 bp AQ17 Number of reads 100 bp or longer at Q17 or betterbased on AQ17 Bases Number of base pairs of sequence from reads with Q17or better

Expanded Reports for Individual Test Fragments:

TABLE 13 The following parameters relating to the performance ofindividual Test Fragments can also be downloaded from the Ion TorrentBrowser: TF Name Test Fragment label. TF Reads Number of reads that areassociated with this Test Fragment. TF Key Measure of strength of thevoltage signal associated with this TF AQ17 Average length of this TestFragment, in base pairs.

Representative results of data obtained from sequencing reactions in theIon Torrent PGM™ sequencing, performed essentially as described aboveand comparing results obtaining the unmodified (reference) Bstpolymerase of SEQ ID NO: 1 and the modified Bst polymerase of SEQ ID NO:2 are shown in FIGS. 3, 4, and 5. FIG. 3 shows the 50Q17 vs. Keypass fora sequencing reaction using the unmodified Bst DNA polymerase having theamino acid sequence of SEQ ID NO: 1 (“manta”) and the modified Bst DNApolymerase having the amino acid sequence of SEQ ID NO: 2 (“ion”).

FIG. 4 shows the 100Q17 vs. Keypass for a sequencing reaction using theunmodified Bst DNA polymerase having the amino acid sequence of SEQ IDNO: 1 (“manta”) and the modified Bst DNA polymerase having the aminoacid sequence of SEQ ID NO: 2 (“ion”).

FIG. 5 shows the Carryforward vs. 50Q17/Keypass for a sequencingreaction using a reference Bst DNA polymerase having the amino acidsequence of SEQ ID NO: 1 (“manta”) and a modified Bst DNA polymerasehaving the amino acid sequence of SEQ ID NO: 2 (“ion”).

The raw data obtained for the sequencing run is depicted in FIG. 6. “IE”refers to the numbers of Incomplete Extensions observed in eachreaction; “CF” refers to the number of carryforwards observed in eachreaction.

TABLE 2 Candidate amino acid residues for modification in Bst DNApolymerase (including pKa values for amino acid residues calculatedusing PropKa) Amino Acid Residue pKa GLU-277 4.64 GLU-372 4.7 GLU-154.71 GLU-206 4.71 GLU-426 4.71 GLU-493 4.71 GLU-456 4.76 GLU-131 4.78GLU-349 4.78 GLU-446 4.85 GLU-522 4.85 GLU-558 4.85 GLU-26 4.92 GLU-2944.92 GLU-363 5.08 HIS-534 5.12 HIS-572 6.17 S HIS-473 6.29 B HIS-46 6.43S HIS-273 6.51 S LYS-510 7.29 B N+ 7.86 S TYR-477 7.98 B LYS-73 8.55CYS-550 8.87 CYS-93 9.57

TABLE 3 Candidate amino acid residues for modification in Bst DNApolymerase (including pKa values for amino acid residues calculatedusing H++) Amino Acid Residue pKa GLU-461 4.533 GLU-206 4.555 ASP-1134.57 HIS-273 4.662 HIS-534 4.747 GLU-277 4.806 GLU-456 4.902 GLU-5444.972 GLU-54 5.037 GLU-30 5.149 GLU-349 5.158 GLU-522 5.221 HIS-151 5.26GLU-558 5.361 GLU-220 5.423 GLU-446 5.911 S NTA 6.465 S HIS-46 7.653 SHIS-572 8.056 S HIS-308 8.333 HIS-473 8.397 LYS-411 9.494 LYS-543 9.72LYS-287 9.815 TYR-419 10.041 LYS-253 10.077

TABLE 4 Candidate amino acids for modification in E. coli SSB Amino AcidResidue pKa HIS-55 3.968 ASP-90 4.251 ASP-17 4.298 ASP-42 4.449 GLU-504.738 GLU-65 4.856 GLU-47 4.878 GLU-19 4.896 GLU-53 5.046 ASP-95 5.143GLU-69 5.214 GLU-38 5.606 NTALA-1 5.851 GLU-80 5.898 LYS-7 7.276 LYS-498.791 GLU-100 9.033 ARG-3 9.069 LYS-87 9.453 LYS-62 9.754 LYS-43 10.399TYR-22 10.427 TYR-97 10.483 TYR-70 10.619 LYS-73 10.898 ARG-56 11.169ARG-96 11.176 ARG-86 11.257 ARG-84 11.296 ARG-41 11.381 ARG-115 11.804ARG-21 12.035 ARG-72 12.671 TYR-78 16.412

TABLE 5 Candidate amino acids for substitution in Therminator ™ DNApolymerase Amino Acid pKa pKa Residue (calc) (model) ASP 4 7.85 ASP 65.95 ASP 31 2.11 ASP 44 2.53 ASP 45 3.60 ASP 50 2.82 ASP 92 4.14 ASP 983.43 ASP 108 4.05 3.80 ASP 113 3.10 3.80 ASP 123 4.09 3.80 ASP 132 3.643.80 ASP 164 1.56 3.80 ASP 177 3.87 3.80 ASP 182 3.79 3.80 ASP 202 3.473.80 ASP 204 2.82 3.80 ASP 212 2.14 3.80 ASP 215 7.23 3.80 ASP 235 2.593.80 ASP 246 3.29 3.80 ASP 259 6.50 3.80 ASP 315 5.97 3.80 ASP 343 3.693.80 ASP 373 3.18 3.80 ASP 398 4.52 3.80 ASP 404 6.10 3.80 ASP 421 4.423.80 ASP 432 2.60 3.80 ASP 444 3.20 3.80 ASP 455 3.97 3.80 ASP 472 3.033.80 ASP 480 3.21 3.80 ASP 540 3.92 3.80 ASP 542 4.89 3.80 ASP 552 2.693.80 ASP 598 3.40 3.80 ASP 614 3.89 3.80 ASP 635 2.78 3.80 ASP 712 3.833.80 ASP 718 3.80 3.80 GLU 10 4.05 4.50 GLU 22 4.12 4.50 GLU 25 3.604.50 GLU 29 4.26 4.50 GLU 35 2.57 4.50 GLU 49 3.85 4.50 GLU 69 4.56 4.50GLU 81 4.38 4.50 GLU 111 4.60 4.50 GLU 130 4.51 4.50 GLU 133 3.91 4.50GLU 134 4.91 4.50 GLU 148 5.27 4.50 GLU 150 5.08 4.50 GLU 151 4.10 4.50GLU 167 4.42 4.50 GLU 187 4.70 4.50 GLU 189 3.57 4.50 GLU 200 4.18 4.50GLU 224 4.29 4.50 GLU 225 4.55 4.50 GLU 238 4.49 4.50 GLU 251 4.60 4.50GLU 276 4.43 4.50 GLU 280 4.75 4.50 GLU 288 3.94 4.50 GLU 293 4.71 4.50GLU 294 3.67 4.50 GLU 300 4.26 4.50 GLU 303 4.59 4.50 GLU 306 4.72 4.50GLU 314 5.00 4.50 GLU 321 4.64 4.50 GLU 325 4.87 4.50 GLU 330 7.31 4.50GLU 354 5.67 4.50 GLU 366 6.07 4.50 GLU 374 4.64 4.50 GLU 376 3.89 4.50GLU 391 4.65 4.50 GLU 393 3.42 4.50 GLU 426 4.46 4.50 GLU 430 4.75 4.50GLU 436 4.54 4.50 GLU 458 4.49 4.50 GLU 459 3.84 4.50 GLU 475 4.11 4.50GLU 508 4.65 4.50 GLU 511 3.78 4.50 GLU 519 4.91 4.50 GLU 522 4.09 4.50GLU 527 2.97 4.50 GLU 529 4.67 4.50 GLU 530 4.53 4.50 GLU 554 4.84 4.50GLU 562 4.46 4.50 GLU 576 5.03 4.50 GLU 578 3.64 4.50 GLU 580 4.99 4.50GLU 599 5.35 4.50 GLU 600 6.04 4.50 GLU 609 5.62 4.50 GLU 617 4.45 4.50GLU 621 4.96 4.50 GLU 628 4.02 4.50 GLU 637 5.22 4.50 GLU 638 4.75 4.50GLU 645 4.50 4.50 GLU 664 4.36 4.50 GLU 719 4.28 4.50 GLU 730 4.72 4.50GLU 734 4.98 4.50 GLU 742 3.65 4.50 C-750 3.25 3.20 HIS 59 6.13 6.50 HIS89 4.69 6.50 HIS 103 7.00 6.50 HIS 147 7.17 6.50 HIS 257 4.01 6.50 HIS416 5.54 6.50 HIS 439 6.77 6.50 HIS 545 2.90 6.50 HIS 633 6.96 6.50 HIS663 5.84 6.50 HIS 679 6.64 6.50 CYS 223 11.84 9.00 CYS 428 99.99 99.99CYS 442 99.99 99.99 CYS 506 99.99 99.99 CYS 509 99.99 99.99 TYR 7 10.5910.00 TYR 30 10.26 10.00 TYR 37 17.23 10.00 TYR 39 14.20 10.00 TYR 8610.20 10.00 TYR 110 11.95 10.00 TYR 112 10.49 10.00 TYR 120 13.12 10.00TYR 146 11.21 10.00 TYR 162 11.82 10.00 TYR 180 11.47 10.00 TYR 20913.47 10.00 TYR 218 11.91 10.00 TYR 261 10.23 10.00 TYR 273 9.77 10.00TYR 279 11.96 10.00 TYR 291 10.52 10.00 TYR 311 14.21 10.00 TYR 32010.89 10.00 TYR 362 11.49 10.00 TYR 384 11.17 10.00 TYR 388 12.23 10.00TYR 402 14.32 10.00 TYR 409 14.74 10.00 TYR 431 10.05 10.00 TYR 48110.52 10.00 TYR 494 12.59 10.00 TYR 496 16.00 10.00 TYR 497 14.40 10.00TYR 499 11.49 10.00 TYR 505 11.27 10.00 TYR 520 11.38 10.00 TYR 53813.52 10.00 TYR 566 11.47 10.00 TYR 579 11.62 10.00 TYR 583 11.55 10.00TYR 594 12.23 10.00 TYR 701 14.24 10.00 TYR 731 10.99 10.00 TYR 73211.88 10.00 TYR 750 10.81 10.00 LYS 13 10.18 10.50 LYS 20 11.10 10.50LYS 21 10.56 10.50 LYS 27 11.24 10.50 LYS 43 10.25 10.50 LYS 52 11.0810.50 LYS 53 10.63 10.50 LYS 57 10.28 10.50 LYS 64 10.26 10.50 LYS 6610.51 10.50 LYS 70 11.41 10.50 LYS 73 9.02 10.50 LYS 74 10.47 10.50 LYS84 10.64 10.50 LYS 118 10.50 10.50 LYS 124 10.20 10.50 LYS 174 10.0110.50 LYS 175 10.42 10.50 LYS 188 10.15 10.50 LYS 192 11.36 10.50 LYS201 12.01 10.50 LYS 220 10.65 10.50 LYS 221 10.80 10.50 LYS 229 10.5410.50 LYS 240 10.09 10.50 LYS 253 11.39 10.50 LYS 285 10.22 10.50 LYS287 11.74 10.50 LYS 289 9.16 10.50 LYS 317 10.31 10.50 LYS 360 8.9110.50 LYS 363 10.27 10.50 LYS 371 9.91 10.50 LYS 390 10.38 10.50 LYS 42910.64 10.50 LYS 440 10.41 10.50 LYS 443 10.92 10.50 LYS 462 11.52 10.50LYS 464 8.83 10.50 LYS 466 10.69 10.50 LYS 468 10.07 10.50 LYS 476 10.2610.50 LYS 477 10.91 10.50 LYS 487 10.75 10.50 LYS 501 10.22 10.50 LYS507 10.64 10.50 LYS 531 11.77 10.50 LYS 535 10.87 10.50 LYS 557 10.5110.50 LYS 558 10.76 10.50 LYS 559 10.31 10.50 LYS 561 10.04 10.50 LYS565 10.25 10.50 LYS 591 10.45 10.50 LYS 592 10.20 10.50 LYS 593 9.7410.50 LYS 602 10.54 10.50 LYS 620 10.49 10.50 LYS 632 10.36 10.50 LYS644 10.24 10.50 LYS 684 10.40 10.50 LYS 692 10.25 10.50 LYS 705 9.5710.50 LYS 746 11.49 10.50 ARG 17 12.25 12.50 ARG 32 13.03 12.50 ARG 5812.29 12.50 ARG 67 14.10 12.50 ARG 78 12.36 12.50 ARG 97 11.98 12.50 ARG99 12.16 12.50 ARG 101 14.07 12.50 ARG 119 16.80 12.50 ARG 169 13.5512.50 ARG 193 12.78 12.50 ARG 196 12.44 12.50 ARG 199 12.34 12.50 ARG222 13.63 12.50 ARG 234 12.80 12.50 ARG 243 12.36 12.50 ARG 247 12.2712.50 ARG 255 10.00 12.50 ARG 265 13.14 12.50 ARG 266 11.19 12.50 ARG307 12.83 12.50 ARG 310 13.21 12.50 ARG 324 12.66 12.50 ARG 335 12.1612.50 ARG 346 13.10 12.50 ARG 359 10.29 12.50 ARG 364 11.85 12.50 ARG375 12.65 12.50 ARG 379 12.22 12.50 ARG 380 12.33 12.50 ARG 381 12.4412.50 ARG 394 12.45 12.50 ARG 406 12.99 12.50 ARG 425 11.45 12.50 ARG460 11.44 12.50 ARG 465 12.42 12.50 ARG 482 10.68 12.50 ARG 484 12.3112.50 ARG 503 13.11 12.50 ARG 518 13.62 12.50 ARG 526 12.48 12.50 ARG585 12.11 12.50 ARG 606 13.59 12.50 ARG 612 12.73 12.50 ARG 613 12.4612.50 ARG 625 12.48 12.50 ARG 641 12.85 12.50 ARG 685 13.07 12.50 ARG689 13.04 12.50 ARG 694 12.46 12.50 ARG 713 12.20 12.50 ARG 743 12.3012.50 N + 1 7.38 8.00

TABLE 6 Candidate amino acids for substitution in KOD DNA polymeraseAmino Acid pKa pKa Residue (calc) (model) ASP 4 7.06 3.80 ASP 6 −1.723.80 ASP 11 3.94 3.80 ASP 31 1.79 3.80 ASP 44 3.94 3.80 ASP 45 2.58 3.80ASP 92 2.18 3.80 ASP 98 3.41 3.80 ASP 108 3.11 3.80 ASP 113 −0.42 3.80ASP 123 1.89 3.80 ASP 132 3.23 3.80 ASP 141 15.80 3.80 ASP 164 3.36 3.80ASP 177 3.87 3.80 ASP 182 3.31 3.80 ASP 202 −1.29 3.80 ASP 204 1.17 3.80ASP 212 −3.07 3.80 ASP 215 4.76 3.80 ASP 235 −0.47 3.80 ASP 246 4.013.80 ASP 259 4.82 3.80 ASP 315 3.17 3.80 ASP 343 3.50 3.80 ASP 373 2.823.80 ASP 404 3.03 3.80 ASP 421 3.55 3.80 ASP 432 3.68 3.80 ASP 444 3.153.80 ASP 455 3.74 3.80 ASP 472 2.98 3.80 ASP 480 3.62 3.80 ASP 540 3.093.80 ASP 542 8.78 3.80 ASP 552 3.53 3.80 ASP 598 3.43 3.80 ASP 614 −1.003.80 ASP 633 3.59 3.80 ASP 635 3.18 3.80 ASP 718 −2.60 3.80 ASP 721−4.20 3.80 ASP 728 −5.80 3.80 ASP 754 −7.40 3.80 GLU 10 4.03 4.50 GLU 223.47 4.50 GLU 25 4.06 4.50 GLU 29 3.91 4.50 GLU 35 3.43 4.50 GLU 49 3.794.50 GLU 50 4.50 4.50 GLU 57 4.71 4.50 GLU 69 3.54 4.50 GLU 81 3.98 4.50GLU 102 4.78 4.50 GLU 111 2.22 4.50 GLU 130 4.78 4.50 GLU 133 4.36 4.50GLU 134 4.50 4.50 GLU 143 9.42 4.50 GLU 148 4.64 4.50 GLU 150 5.76 4.50GLU 151 15.70 4.50 GLU 154 4.10 4.50 GLU 165 3.26 4.50 GLU 166 4.35 4.50GLU 187 0.05 4.50 GLU 189 4.21 4.50 GLU 200 4.43 4.50 GLU 224 4.64 4.50GLU 238 3.49 4.50 GLU 251 4.31 4.50 GLU 276 0.56 4.50 GLU 280 4.78 4.50GLU 288 4.61 4.50 GLU 293 4.50 4.50 GLU 294 3.98 4.50 GLU 300 4.85 4.50GLU 303 4.57 4.50 GLU 306 4.69 4.50 GLU 314 3.66 4.50 GLU 321 3.37 4.50GLU 325 4.50 4.50 GLU 330 6.95 4.50 GLU 354 3.49 4.50 GLU 363 1.46 4.50GLU 366 4.66 4.50 GLU 374 4.36 4.50 GLU 376 3.83 4.50 GLU 385 4.50 4.50GLU 391 4.64 4.50 GLU 393 3.84 4.50 GLU 398 3.91 4.50 GLU 426 2.90 4.50GLU 430 4.57 4.50 GLU 458 4.50 4.50 GLU 459 4.53 4.50 GLU 475 4.54 4.50GLU 508 3.90 4.50 GLU 511 4.19 4.50 GLU 519 4.57 4.50 GLU 527 4.04 4.50GLU 529 3.93 4.50 GLU 530 4.05 4.50 GLU 554 4.60 4.50 GLU 562 4.64 4.50GLU 576 4.47 4.50 GLU 578 5.53 4.50 GLU 580 4.12 4.50 GLU 599 4.05 4.50GLU 600 4.64 4.50 GLU 609 14.10 4.50 GLU 617 12.50 4.50 GLU 621 10.904.50 GLU 628 4.47 4.50 GLU 637 4.50 4.50 GLU 645 4.50 4.50 GLU 648 4.504.50 GLU 654 4.50 4.50 GLU 658 3.94 4.50 GLU 664 9.30 4.50 GLU 719 7.704.50 GLU 730 6.10 4.50 GLU 734 4.50 4.50 GLU 742 2.90 4.50 GLU 753 1.304.50 HIS 59 6.43 6.50 HIS 89 4.54 6.50 HIS 103 6.15 6.50 HIS 147 6.366.50 HIS 257 4.00 6.50 HIS 416 2.74 6.50 HIS 439 7.09 6.50 HIS 663 6.506.50 HIS 679 6.50 6.50 HIS 725 6.50 6.50 CYS 223 10.07 9.00 CYS 42899.99 9.00 CYS 442 99.99 9.00 CYS 506 6.50 9.00 CYS 509 16.78 9.00 TYR 710.67 10.00 TYR 30 10.00 10.00 TYR 37 17.98 10.00 TYR 39 12.92 10.00 TYR86 13.65 10.00 TYR 110 12.67 10.00 TYR 112 10.84 10.00 TYR 120 13.5310.00 TYR 146 10.06 10.00 TYR 162 10.41 10.00 TYR 180 10.00 10.00 TYR209 11.28 10.00 TYR 218 7.75 10.00 TYR 261 9.34 10.00 TYR 273 9.22 10.00TYR 279 13.15 10.00 TYR 291 10.00 10.00 TYR 311 16.84 10.00 TYR 32014.28 10.00 TYR 362 12.68 10.00 TYR 384 10.00 10.00 TYR 388 10.00 10.00TYR 402 17.93 10.00 TYR 409 12.25 10.00 TYR 431 9.81 10.00 TYR 481 11.7610.00 TYR 493 12.60 10.00 TYR 494 9.46 10.00 TYR 496 14.11 10.00 TYR 49715.66 10.00 TYR 499 9.84 10.00 TYR 505 8.20 10.00 TYR 520 11.19 10.00TYR 532 11.04 10.00 TYR 538 12.70 10.00 TYR 566 13.34 10.00 TYR 57910.65 10.00 TYR 583 13.57 10.00 TYR 594 10.60 10.00 TYR 653 9.87 10.00TYR 701 10.00 10.00 TYR 750 10.24 10.00 N+ 17.37 8.00 LYS 13 10.15 10.50LYS 20 10.22 10.50 LYS 21 9.87 10.50 LYS 27 10.36 10.50 LYS 43 10.4310.50 LYS 52 10.08 10.50 LYS 53 10.08 10.50 LYS 66 10.01 10.50 LYS 709.94 10.50 LYS 73 10.06 10.50 LYS 74 10.50 10.50 LYS 84 9.60 10.50 LYS99 10.50 10.50 LYS 118 11.22 10.50 LYS 124 9.94 10.50 LYS 174 10.4310.50 LYS 192 10.36 10.50 LYS 199 8.13 10.50 LYS 201 9.94 10.50 LYS 22010.29 10.50 LYS 221 10.22 10.50 LYS 225 10.50 10.50 LYS 240 10.01 10.50LYS 253 12.95 10.50 LYS 287 14.68 10.50 LYS 289 11.48 10.50 LYS 31710.23 10.50 LYS 324 10.08 10.50 LYS 360 11.55 10.50 LYS 371 9.51 10.50LYS 375 10.50 10.50 LYS 429 10.50 10.50 LYS 443 10.22 10.50 LYS 46210.50 10.50 LYS 466 10.22 10.50 LYS 468 10.29 10.50 LYS 477 10.50 10.50LYS 487 10.24 10.50 LYS 507 11.91 10.50 LYS 526 10.22 10.50 LYS 53110.15 10.50 LYS 535 10.36 10.50 LYS 557 10.01 10.50 LYS 565 10.50 10.50LYS 570 10.36 10.50 LYS 592 10.50 10.50 LYS 602 10.43 10.50 LYS 63210.36 10.50 LYS 638 10.15 10.50 LYS 726 9.87 10.50 ARG 17 15.49 12.50ARG 32 12.15 12.50 ARG 58 11.45 12.50 ARG 67 11.73 12.50 ARG 78 11.9412.50 ARG 97 12.43 12.50 ARG 101 12.08 12.50 ARG 119 17.00 12.50 ARG 16912.15 12.50 ARG 188 11.94 12.50 ARG 193 13.69 12.50 ARG 196 12.29 12.50ARG 222 14.49 12.50 ARG 234 14.17 12.50 ARG 243 12.50 12.50 ARG 24712.01 12.50 ARG 255 9.85 12.50 ARG 265 12.01 12.50 ARG 266 10.80 12.50ARG 307 12.15 12.50 ARG 310 12.22 12.50 ARG 335 8.74 12.50 ARG 346 11.1612.50 ARG 359 9.85 12.50 ARG 364 11.90 12.50 ARG 379 12.08 12.50 ARG 38011.96 12.50 ARG 381 12.15 12.50 ARG 394 12.50 12.50 ARG 406 12.39 12.50ARG 425 11.45 12.50 ARG 440 12.50 12.50 ARG 460 11.45 12.50 ARG 47612.22 12.50 ARG 482 11.15 12.50 ARG 484 12.22 12.50 ARG 501 12.22 12.50ARG 503 13.21 12.50 ARG 518 11.94 12.50 ARG 585 11.66 12.50 ARG 60612.29 12.50 ARG 612 12.50 12.50 ARG 613 12.50 12.50 ARG 625 12.29 12.50ARG 641 12.15 12.50 ARG 685 12.50 12.50 ARG 713 12.50 12.50 ARG 74312.50 12.50 ARG 746 11.94 12.50 ARG 751 12.50 12.50 ARG 756 12.01 12.50

TABLE 7 Candidate amino acids for modification in B103-type polymerasesAmino Acid Substituted Amino Acid Substituted Residue Amino Acid ResidueAmino Acid H58 R E290 A H73 R E293 A H74 R E311 A H103 R E319 A H146 RE322 A H153 R E331 A H336 R E335 A H370 R E338 A H458 R E343 A H482 RE352 A E11 A E28 A E359 A E43 A E371 A E50 A E405 A E72 A E416 A E81 AE417 A E148 A E463 A E154 A E466 A E158 A E483 A E159 A E505 A E161 AE512 A E168 A E517 A E216 A C7 S E236 A C19 S E238 A C103 S E241 A C445S E273 A C452 S E276 A C513 S E288 A C527 S

What is claimed:
 1. A modified Bst DNA polymerase comprising three ormore amino acid substitutions selected from the group consisting ofH46R, H273R, H281R, E446Q, H473R, Y477F, H528R, and H572R, the numberingof amino acid residues being in accordance with that of SEQ ID NO: 1,wherein the modified polymerase has reduced buffering capacity comparedto wild type Bst DNA polymerase, retains the ability to catalyzenucleotide polymerization, and comprises an amino acid sequence havingat least 90% identity to the amino acid sequence of SEQ ID NO:
 1. 2. Themodified Bst DNA polymerase of claim 1, wherein the amino acidsubstitutions comprise H46R, E446Q, and H572R.
 3. The modified Bst DNApolymerase of claim 2, further comprising one or more amino acidsubstitutions of one or more amino acid residues shown in Table 2 or 3.4. The modified Bst DNA polymerase of claim 1, wherein the modifiedpolymerase has reduced buffering capacity compared to wild type Bst DNApolymerase within a range of about pH 4 to about pH
 10. 5. The modifiedBst DNA polymerase of claim 1, wherein the modified polymerase hasreduced buffering capacity compared to wild type Bst DNA polymerasewithin a range of about pH 7 to about pH
 9. 6. The modified Bst DNApolymerase of claim 1, wherein polymerase activity of the modified BstDNA polymerase is at least 90% compared to wild type Bst DNA polymerase.7. An aqueous solution comprising the modified Bst DNA polymerase ofclaim 1, wherein said aqueous solution further comprises a templatenucleic acid and nucleotides.
 8. A method of detecting a nucleotideincorporation, comprising: (a) performing a nucleotide incorporation bycontacting a template nucleic acid with the modified Bst DNA polymeraseof claim 1 in the presence of nucleotides, wherein one or more hydrogenions are generated as a by-product of the nucleotide incorporation; and(b) detecting the one or more hydrogen ions generated as a by-product ofthe nucleotide incorporation, thereby detecting the nucleotideincorporation.
 9. The method of claim 8, wherein the amino acidsubstitutions comprise H46R, E446Q, and H572R.
 10. The method of claim9, wherein the modified Bst DNA polymerase further comprises one or moreamino acid substitutions of one or more amino acid residues shown inTable 2 or
 3. 11. The method of claim 8, wherein the modified Bst DNApolymerase has reduced buffering capacity compared to wild type Bst DNApolymerase within a range of about pH 4 to about pH
 10. 12. The methodof claim 8, wherein the modified Bst DNA polymerase has reducedbuffering capacity compared to wild type Bst DNA polymerase within arange of about pH 7 to about pH
 9. 13. The method of claim 8, whereinthe buffering capacity of the modified Bst DNA polymerase between pH 7and pH 9 has been removed.
 14. A method of sequencing a nucleic acid,comprising: (a) contacting a template nucleic acid with the modified BstDNA polymerase of claim 1 and nucleotides; (b) synthesizing a newnucleic acid strand by sequentially incorporating one or more of thenucleotides into a nucleic acid molecule; and (c) detecting suchincorporation by detecting one or more hydrogen ion byproducts generatedwhen the nucleotide is complementary to a corresponding nucleotide inthe template nucleic acid.
 15. The method of claim 14, wherein the aminoacid substitutions comprise H46R, E446Q, and H572R.
 16. The method ofclaim 15, wherein the modified Bst DNA polymerase further comprises oneor more amino acid substitutions of one or more amino acid residuesshown in Table 2 or
 3. 17. The method of claim 14, wherein the modifiedBst DNA polymerase has reduced buffering capacity compared to wild typeBst DNA polymerase within a range of about pH 4 to about pH
 10. 18. Themethod of claim 14, wherein the modified Bst DNA polymerase has reducedbuffering capacity compared to wild type Bst DNA polymerase within arange of about pH 7 to about pH
 9. 19. The method of claim 14, whereinthe buffering capacity of the modified Bst DNA polymerase between pH 7and pH 9 has been removed.
 20. The method of claim 14, wherein thetemplate nucleic acid is hybridized to a sequencing primer whencontacting the modified Bst DNA polymerase and synthesizing the newnucleic acid strand by incorporating the one or more nucleotides at the3′ end of the sequencing primer.